Casein hydrolysate capable of improving xanthine oxidase inhibitory activity and preparation method of casein hydrolysate
By optimizing casein hydrolysate through ultrasonic treatment and alkaline protease hydrolysis, the problem of insufficient xanthine oxidase inhibitory activity in casein hydrolysate was solved, resulting in a significant improvement in the bioactivity of casein hydrolysate, making it suitable for the preparation of health products for treating hyperuricemia.
Patent Information
- Application Number
- CN202511303764.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-12
- Publication Date
- 2025-12-12
AI Technical Summary
Existing enzymatic hydrolysis techniques are insufficient to significantly improve the xanthine oxidase inhibitory activity of casein hydrolysates, and traditional methods have limited hydrolysis degree, making it difficult to enhance biological activity.
Casein was physically modified using ultrasonic treatment technology, combined with alkaline protease hydrolysis, and the ultrasonic power and time were optimized to improve the xanthine oxidase inhibitory activity of casein hydrolysate.
It significantly increases the content of small molecule active substances and xanthine oxidase inhibitory activity in casein hydrolysate, enhances biological activity, and is suitable for preparing health products for treating hyperuricemia.
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Figure CN121109538A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of bioactive peptide preparation technology, specifically to a casein hydrolysate with enhanced xanthine oxidase inhibitory activity and its preparation method. Background Technology
[0002] Hyperuricemia is a metabolic disease caused by purine metabolism disorders or decreased uric acid excretion, characterized by elevated serum uric acid levels. This condition is not only a major risk factor for gout but is also closely related to various other diseases such as diabetes, chronic kidney disease, and hypertension. Xanthine oxidase (XO) is a key enzyme in the purine metabolism pathway, catalyzing the oxidation of hypoxanthine and xanthine to uric acid, while simultaneously producing superoxide anions (O₂). 2– Hydrogen peroxide (H2O2) and reactive oxygen species (ROS) are active substances in the body. Currently, the clinical management of hyperuricemia still faces many challenges, and inhibiting XO activity has been considered an important strategy for lowering uric acid levels and alleviating oxidative stress. Commonly used XO inhibitors include allopurinol and febuxo, which are effective in treating gout, but long-term use can lead to significant side effects. Therefore, finding safe, effective, and non-toxic XO inhibitors from natural active substances has become a feasible strategy for managing hyperuricemia.
[0003] Bioactive peptides have attracted much attention due to their potential anti-hyperuricemia activity. Multiple studies have shown that short peptides are more easily absorbed by the human body and exhibit stronger XO inhibitory activity. Casein-derived peptides, as an important source of milk-derived bioactive peptides, are effective functional components for inhibiting XO activity. However, traditional enzymatic hydrolysis techniques have limited hydrolysis degrees, making it difficult to enhance the overall bioactivity of hydrolysates by increasing the content of specific bioactive peptides. Therefore, auxiliary or combined methods are needed to improve the bioactivity of casein hydrolysates. Ultrasound, as an emerging technology, can act on substrates or enzymes and is an effective method to increase the protein hydrolysis rate or enhance bioactivity. Therefore, this invention uses casein as a raw material and utilizes ultrasonic treatment technology to physically modify it, aiming to obtain casein peptides with high xanthine oxidase inhibitory activity. Summary of the Invention
[0004] To overcome the shortcomings of existing technologies, the purpose of this invention is to provide a casein hydrolysate with enhanced xanthine oxidase inhibitory activity and its preparation method. The preparation method provided by this invention is simple, and the entire process meets food-grade requirements, making it applicable to pharmaceuticals, health products, and food industries.
[0005] This invention provides a casein hydrolysate with enhanced xanthine oxidase inhibitory activity and its preparation method, the specific steps of which are as follows:
[0006] (1) Casein was pretreated with ultrasonic power of 150W, 300W, 450W and 600W for 15 min respectively; (2) The sample prepared in step (1) was hydrolyzed with alkaline protease for 3 h. After hydrolysis, the enzyme was heated to inactivate the enzyme, then cooled to room temperature, and then frozen at -20℃ for subsequent analysis; (3) The surface hydrophobicity of the sample prepared in step (1) was measured; (4) The degree of hydrolysis of the sample prepared in step (2) was measured; (5) The relative molecular weight distribution of the sample prepared in step (2) was measured; (6) The xanthine oxidase inhibitory activity of the sample prepared in step (2) was measured; (7) The ROS level of the sample prepared in step (2) was measured.
[0007] The ultrasonic conditions for step (1) are: ultrasonication for 15 minutes under ultrasonic power of 150W, 300W, 450W, and 600W.
[0008] In step (2), the casein concentration is 5%, the hydrolysis time is 3 hours, and the enzyme-to-protein ratio is 4500 U / g.
[0009] The optimal ultrasonic treatment conditions for steps (4) and (6) are 450W;
[0010] The method for detecting xanthine oxidase inhibitory activity in step (6) is as follows: Take 50 μL of sample and 50 μL of xanthine oxidase solution and react them at 37°C in the dark for 30 min. Then add 50 μL of xanthine solution (prepared using 50 mmol / L PBS solution to a concentration of 0.7 mmol / L) and react at 37°C in the dark for 5 min. Measure the absorbance at 295 nm using a microplate reader. The formula for calculating xanthine oxidase inhibitory activity (IR) is as follows.
[0011]
[0012] In the formula, A1 (sample, XO, XA), A2 (XO, PBS, XA), A3 (sample, PBS, XA), and A4 (PBS).
[0013] Compared with the prior art, the advantages of this invention are:
[0014] The casein hydrolysate provided by this invention, which enhances the inhibitory activity of xanthine oxidase, is a casein hydrolysate containing small molecule active substances. It effectively enhances the inhibitory activity of xanthine oxidase, with the content of <3kDa in the hydrolysate increasing by 5.75% and the inhibitory activity of xanthine oxidase increasing by 9.04%.
[0015] The casein hydrolysate provided by this invention has enhanced xanthine oxidase inhibitory activity. In vitro verification has shown that it has high xanthine oxidase inhibitory activity and can be used to prepare health products that enhance anti-hyperuricemia. Attached Figure Description
[0016] Figure 1 This is a process flow diagram of the present invention;
[0017] Figure 2 The surface hydrophobicity of casein under different ultrasonic powers;
[0018] Figure 3 The effect of ultrasonic power on the degree of hydrolysis of casein hydrolysate;
[0019] Figure 4 The effect of ultrasonic power on the molecular weight of casein hydrolysate;
[0020] Figure 5 The effect of ultrasonic power on the xanthine oxidase inhibitory activity of casein hydrolysate;
[0021] Figure 6 This relates to the effect of ultrasonic power on the ROS level of casein hydrolysate. Detailed Implementation
[0022] The specific embodiments are further described below with reference to the accompanying drawings.
[0023] A method for preparing casein anti-anxiety peptides, characterized in that the method includes the following steps:
[0024] Example 1
[0025] (1) Using the controlled variable method, 5% casein was treated with an ultrasonic cell disruptor at various ultrasonic powers of 150W for 15 minutes. The ultrasonic mode was set to a frequency of 20kHz, with a pulse cycle of 2s on / 2s off. The sample temperature was kept below 15℃ in an ice-water bath. The untreated sample was named 0W, and the treated sample was named 150W according to the power.
[0026] (2) Casein was pretreated by ultrasound, alkaline protease was selected, enzyme-to-protein ratio was 4500 U / g, and casein was hydrolyzed for 3 hours. 10 mL samples were taken at intervals and the enzyme was inactivated in a 95℃ water bath for 10 min. The samples were then frozen and stored for subsequent analysis.
[0027] (3) After diluting the sample prepared in step (1) by 100 times, the surface hydrophobicity is measured, such as... Figure 2 As shown, under ultrasonic power of 150W, the surface hydrophobicity was improved by 8.41% compared with no ultrasonic treatment. This is attributed to the ultrasonic pretreatment causing the casein structure to unfold and expose the hydrophobic regions hidden inside.
[0028] (4) Dilute the sample prepared in step (2) 300 times and determine the degree of hydrolysis. Figure 3As shown, the degree of hydrolysis was 16.83% under ultrasonic power of 150W, which was 1.41% higher than that without ultrasonication. This is attributed to the strong shearing force of ultrasound promoting protein structure unfolding, exposing more enzyme cleavage sites, and thus improving enzyme cleavage efficiency.
[0029] (5) The sample prepared in step (2) was diluted 10 times, filtered through a 0.22 μm microporous membrane, and the molecular weight content was determined. The results are shown in Table 1.
[0030]
[0031] Analysis of the results in Table 1 shows that, at an ultrasonic power of 150W, the content of small molecule peptides <3kDa increased by 2.41%.
[0032] (6) Dilute the sample prepared in step (2) 10 times and determine the xanthine oxidase inhibitory activity, such as Figure 5 As shown, under ultrasound power of 150W, xanthine oxidase inhibitory activity increased by 5.24% compared with no ultrasound.
[0033] Example 2
[0034] (1) Using the controlled variable method, 5% casein was treated with an ultrasonic cell disruptor at various ultrasonic powers of 300W for 15 minutes. The ultrasonic mode was set to a frequency of 20kHz, with a pulse cycle of 2s on / 2s off. The sample temperature was kept below 15℃ in an ice-water bath. The untreated sample was named 0W, and the treated sample was named 300W according to the power.
[0035] (2) Casein was pretreated by ultrasound, alkaline protease was selected, enzyme-to-protein ratio was 4500 U / g, and casein was hydrolyzed for 3 hours. 10 mL samples were taken at intervals and the enzyme was inactivated in a 95℃ water bath for 10 min. The samples were then frozen and stored for subsequent analysis.
[0036] (3) After diluting the sample prepared in step (1) by 100 times, the surface hydrophobicity is measured, such as... Figure 2 As shown, under ultrasonic power of 300W, the surface hydrophobicity was improved by 24.61% compared with no ultrasonic treatment. This is attributed to the ultrasonic pretreatment causing the casein structure to unfold and expose the hydrophobic regions hidden inside.
[0037] (4) Dilute the sample prepared in step (2) 300 times and determine the degree of hydrolysis. Figure 3 As shown, the degree of hydrolysis was 17.13% under ultrasonic power of 300W, which was 1.71% higher than that without ultrasonication. This is attributed to the strong shearing force of ultrasound promoting protein structure unfolding, exposing more enzyme cleavage sites, and thus improving enzyme cleavage efficiency.
[0038] (5) The sample prepared in step (2) was diluted 10 times, filtered through a 0.22 μm microporous membrane, and the molecular weight content was determined. The results are shown in Table 2.
[0039]
[0040]
[0041] Analysis of the results in Table 2 shows that, at an ultrasonic power of 150W, the content of small molecule peptides <3kDa increased by 4.55%.
[0042] (6) Dilute the sample prepared in step (2) 10 times and determine the xanthine oxidase inhibitory activity, such as Figure 5 As shown, under ultrasound power of 150W, xanthine oxidase inhibitory activity increased by 9.04% compared with no ultrasound.
[0043] Example 3
[0044] (1) Using the controlled variable method, 5% casein was treated with an ultrasonic cell disruptor at various ultrasonic powers of 450W for 15 minutes. The ultrasonic mode was set to a frequency of 20kHz, with a pulse cycle of 2s on / 2s off. The sample temperature was kept below 15℃ in an ice-water bath. The untreated sample was named 0W, and the treated sample was named 150W according to the power.
[0045] (2) Casein was pretreated by ultrasound, alkaline protease was selected, enzyme-to-protein ratio was 4500 U / g, and casein was hydrolyzed for 3 hours. 10 mL samples were taken at intervals and the enzyme was inactivated in a 95℃ water bath for 10 min. The samples were then frozen and stored for subsequent analysis.
[0046] (3) After diluting the sample prepared in step (1) by 100 times, the surface hydrophobicity is measured, such as... Figure 2 As shown, under ultrasonic power of 450W, the surface hydrophobicity was improved by 31.08% compared with no ultrasonic treatment. This is attributed to the ultrasonic pretreatment causing the casein structure to unfold and expose the hydrophobic regions hidden inside.
[0047] (4) Dilute the sample prepared in step (2) 300 times and determine the degree of hydrolysis. Figure 3 As shown, the degree of hydrolysis was 16.83% under ultrasonic power of 450W, which was 4.89% higher than that without ultrasonication. This is attributed to the strong shearing force of ultrasound promoting protein structure unfolding, exposing more enzyme cleavage sites, and thus improving enzyme cleavage efficiency.
[0048] (5) The sample prepared in step (2) was diluted 10 times, filtered through a 0.22 μm microporous membrane, and the molecular weight content was determined. The results are shown in Table 3.
[0049]
[0050] Analysis of the results in Table 3 shows that, at an ultrasonic power of 150W, the content of small molecule peptides <3kDa increased by 5.75%.
[0051] (6) Dilute the sample prepared in step (2) 10 times and determine the xanthine oxidase inhibitory activity, such as Figure 5 As shown, under ultrasonic power of 150W, the xanthine oxidase inhibitory activity increased by 9.04% compared with no ultrasonication.
[0052] (7) Dilute the sample prepared in step (2) to concentrations of 125, 250, and 500 μg / mL, and determine the ROS level, such as Figure 6 As shown.
[0053] Example 4
[0054] (1) Using the controlled variable method, 5% casein was treated with an ultrasonic cell disruptor at various ultrasonic powers of 600W for 15 minutes. The ultrasonic mode was set to a frequency of 20kHz, with a pulse cycle of 2s on / 2s off. The sample temperature was kept below 15℃ in an ice-water bath. The untreated sample was named 0W, and the treated sample was named 600W according to the power.
[0055] (2) Casein was pretreated by ultrasound, alkaline protease was selected, enzyme-to-protein ratio was 4500 U / g, and casein was hydrolyzed for 3 hours. 10 mL samples were taken at intervals and the enzyme was inactivated in a 95℃ water bath for 10 min. The samples were then frozen and stored for subsequent analysis.
[0056] (3) After diluting the sample prepared in step (1) by 100 times, the surface hydrophobicity is measured, such as... Figure 2 As shown, under ultrasonic power of 600W, the surface hydrophobicity was improved by 29.40% compared with no ultrasonic treatment. This is attributed to the ultrasonic pretreatment causing the casein structure to unfold and expose the hydrophobic regions hidden inside.
[0057] (4) Dilute the sample prepared in step (2) 300 times and determine the degree of hydrolysis. Figure 3 As shown, the degree of hydrolysis was 19.54% under ultrasonic power of 600W, which was 4.12% higher than that without ultrasonication. This is attributed to the strong shearing force of ultrasound promoting protein structure unfolding, exposing more enzyme cleavage sites, and thus improving enzyme cleavage efficiency.
[0058] (5) The sample prepared in step (2) was diluted 10 times, filtered through a 0.22 μm microporous membrane, and the molecular weight content was determined. The results are shown in Table 4.
[0059]
[0060]
[0061] The results in Table 4 show that, at an ultrasonic power of 150W, the content of small molecule peptides <3kDa increased by 4.79%.
[0062] (6) Dilute the sample prepared in step (2) 10 times and determine the xanthine oxidase inhibitory activity, such as Figure 5 As shown, under ultrasound power of 600W, xanthine oxidase inhibitory activity increased by 6.11% compared with no ultrasound.
Claims
1. A casein hydrolysate having an increased xanthine oxidase inhibitory activity and a method for preparing the same, characterized by, The method comprises the following steps: (1) setting 150W, 300W, 450W, 600W ultrasonic power respectively to pretreat casein for 15min; (2) hydrolyzing the sample prepared in step (1) by using alkaline protease for 3h, after hydrolysis, the enzyme solution is heated to inactivate the enzyme, then cooled to room temperature, and then stored in-20℃ for freezing preservation, for subsequent analysis; (3) determining the surface hydrophobicity of the sample prepared in step (1); (4) determining the degree of hydrolysis of the sample prepared in step (2); (5) determining the relative molecular weight distribution of the sample prepared in step (2); (6) determining the xanthine oxidase inhibitory activity of the sample prepared in step (2); (7) determining the ROS level of the sample prepared in step (2).
2. The casein hydrolysate having an increased xanthine oxidase inhibitory activity according to claim 1, wherein, The ultrasonic condition is ultrasonic treatment for 15min under the ultrasonic power of 150W, 300W, 450W, 600W.
3. The casein hydrolysate with enhanced xanthine oxidase inhibitory activity according to claim 1, and its preparation method, characterized in that, The casein concentration is 5%, the hydrolysis time is 3h, and the enzyme-substrate ratio is 4500U / g.
4. The casein hydrolysate having an increased xanthine oxidase inhibitory activity according to claim 1, wherein, The optimal ultrasonic treatment condition is 450W.
5. The casein hydrolysate with enhanced xanthine oxidase inhibitory activity according to claim 1, and its preparation method, characterized in that, The detection method of the xanthine oxidase inhibitory activity is: taking 50μL sample and 50μL xanthine oxidase solution to react at 37℃ for 30min in dark, then adding 50μL xanthine solution (prepared by using 50mmol / L PBS solution to be 0.7mmol / L) to react at 37℃ for 5min in dark, using an enzyme label instrument to determine the absorbance at 295nm, and calculating the xanthine oxidase inhibitory activity (IR) according to the following formula: In the formula, A1 (sample, XO, XA), A2 (XO, PBS, XA), A3 (sample, PBS, XA), A4 (PBS).