A bovine colostrum active polypeptide with antioxidant and sleep aid efficacy, and a preparation method and application thereof

Abstract

This application belongs to the field of bovine colostrum peptide technology, and relates to a bovine colostrum active polypeptide with antioxidant and sleep-aiding effects, its preparation method, and its application. The bovine colostrum active polypeptide contains at least one of HQPHQPLPPT, AVPYPQ, HLPIP, VVPPF, YPVEP, or FPPQ. The prepared bovine colostrum active polypeptide exhibits important biological activities: on the one hand, it exerts antioxidant effects by scavenging / inhibiting ABTS free radicals; on the other hand, it inhibits neuronal excitability, regulates the sleep-wake cycle, and improves insomnia by promoting the expression of the inhibitory neurotransmitter GABA and its receptor, as well as 5-hydroxytryptamine synaptic signaling. In terms of flavor, the bovine colostrum active polypeptide has no obvious bitterness or astringency. Furthermore, the preparation method of the bovine colostrum active polypeptide requires low equipment requirements, is simple and easy to operate, and is suitable for large-scale industrial production. This application enriches the bioactive peptide library and is of great significance for promoting technological progress and industrial upgrading in the field of bovine colostrum peptides.

Description

Technical Field

[0001] This application relates to the field of bioactive peptide technology, specifically to an active bovine colostrum polypeptide with antioxidant and sleep-aiding effects, its preparation method, and its application. Background Technology

[0002] Sleep plays a vital role in human health. The fast pace of modern life and work pressure can affect sleep quality, and in severe cases, cause sleep disorders. Insufficient sleep or poor sleep quality can adversely affect multiple systems in the body. Traditional sleep aids typically work through sedation and hypnosis, but they often have significant side effects and potential for addiction, and long-term use may have adverse health effects. Therefore, developing natural, safe, and effective active ingredients with sleep-aiding properties has become a research hotspot.

[0003] Food-derived bioactive peptides have attracted much attention due to their high safety and multiple regulatory effects. Bovine colostrum is rich in nutrients, and the various short peptides with different structures formed after the digestion of proteins in bovine colostrum participate in various physiological functions. However, directly drinking milk has little effect on improving insomnia. Some studies have shown that jujube seed, lily bulb, and bovine milk casein have sedative and sleep-promoting effects. Bovine colostrum peptides are small molecule peptide compounds with specific biological activities released from the core proteins of bovine colostrum through biotechnological methods such as enzymatic hydrolysis or microbial fermentation, and possess a variety of beneficial biological activities. Related studies have found that bovine colostrum is rich in immunoglobulins, growth factors, and antimicrobial peptides, but there is limited research on the sleep-aiding function of bovine colostrum peptides.

[0004] With the deepening research on bioactive peptides, peptides have shown great potential in improving human health due to their high bioactivity, low toxicity, and good targeting properties. Some bioactive peptides can exert physiological effects that promote sleep through mechanisms such as anti-inflammation and regulation of sleep-related hormone levels.

[0005] Therefore, identifying specific fragments in bovine colostrum peptides that have antioxidant and sleep-aiding effects has significant theoretical and practical value. Summary of the Invention

[0006] The purpose of this application is to overcome the shortcomings of the prior art and provide a bovine colostrum active polypeptide with antioxidant and sleep-aiding effects, as well as its preparation method and application.

[0007] This application provides a method for preparing bovine colostrum bioactive peptides, which includes the following steps:

[0008] S1. Mix bovine colostrum protein powder with water evenly and perform shearing to obtain a pretreatment solution. S2. Add lipase to the primary pretreatment solution for secondary pretreatment, and then inactivate the enzyme to obtain a secondary pretreatment mixture. S3. Add Bacillus licheniformis alkaline protease to the secondary pretreatment solution for the first hydrolysis, and then inactivate the enzyme to obtain the first hydrolysate; S4. Add bromelain to the first hydrolysate for a second hydrolysis, and then inactivate the enzyme to obtain the second hydrolysate; S5. The second hydrolysate is subjected to filtration, ultrafiltration and spray drying to obtain powdered bovine colostrum active peptides.

[0009] In some embodiments, in step S1, the mass ratio of bovine colostrum protein powder to water is 1:(8~12); the shearing treatment lasts for 10 to 30 minutes and the temperature is 20°C to 30°C. In step S2, the amount of lipase added is 4.0% to 6.0% of the mass of the bovine colostrum protein powder; the temperature of the secondary pretreatment is 35℃ to 39℃, the reaction pH is 6.5 to 7.5, and the time is 0.5 to 1.5 h.

[0010] In some embodiments, in step S3, the amount of Bacillus licheniformis alkaline protease added is 0.4% to 0.6% of the mass of the bovine colostrum protein powder; in step S4, the amount of bromelain added is 0.8% to 1.2% of the mass of the bovine colostrum protein powder.

[0011] In some embodiments, in step S3, the temperature of the first hydrolysis is 50℃~55℃, the reaction pH is 8.0~9.0, and the hydrolysis time is 1.5~2.5h; in step S4, the temperature of the second hydrolysis is 40℃~50℃, the reaction pH is 6.5~7.0, and the hydrolysis time is 1.5~2.5h.

[0012] In some embodiments, in S2, S3 and S4, the enzyme inactivation conditions are all at 85℃~90℃ for 15~20min; in S5, the ultrafiltration process is performed using an ultrafiltration membrane with a molecular weight cutoff of 5000 Da.

[0013] This application provides a bovine colostrum active polypeptide, which is prepared by the method described above.

[0014] In some embodiments, the bovine colostrum active polypeptide comprises at least one of the peptide sequences HQPHQPLPPT, AVPYPQ, HLPIP, VVPPF, YPVEP, or FPPQ.

[0015] This application also provides a functional product whose active component comprises bovine colostrum active polypeptides as described above.

[0016] This application also provides the use of the bovine colostrum active polypeptide as described above in the preparation of functional products with antioxidant and / or sleep-aiding effects.

[0017] Compared with the prior art, the solution of this application has the following advantages: The bovine colostrum active polypeptide prepared by the method of this application has the effects of inhibiting ABTS, promoting the expression of the inhibitory neurotransmitter GABA and its receptor and 5-hydroxytryptamine synaptic signals, inhibiting neuronal excitability, regulating the sleep-wake cycle, and improving insomnia.

[0018] Furthermore, the bovine colostrum active peptides prepared by the method of this application have no obvious bitter or astringent taste. Moreover, the preparation method of this application has low equipment requirements, is simple and easy to operate, and can obtain the desired bovine colostrum active peptides through a simple combination of operations such as mixing, pretreatment, enzymatic hydrolysis, and filtration, facilitating large-scale industrial production. Attached Figure Description

[0019] The accompanying drawings, which are provided to further illustrate this application and form part of this application, illustrate exemplary embodiments of this application and are used to explain this application, but do not constitute an undue limitation of this application.

[0020] Figure 1 This is a graph showing the data analysis of intracellular γ-aminobutyric acid (GABA) content in PC12 cells during Experiment 2 of this application; Figure 2 This document describes the expression of the GABA gene in PC12 cells during Experiment 2 of this application. Figure 3 This describes the expression of the OX1R gene in PC12 cells during Experiment 2 of this application. Figure 4 This describes the expression of the OX2R gene in PC12 cells during Experiment 2 of this application. Figure 5 For GABA in PC12 cells in Experiment 2 of this application A R gene expression status; Figure 6 This describes the expression of the 5-HT gene in PC12 cells during Experiment 2 of this application. Figure 7 This describes the expression of the TNF-α gene in PC12 cells during Experiment 2 of this application. Figure 8 For the HQPHQPLPPT and GABA in Experiment 3 of this application A Conformation diagram of R and OX2R molecules docking; Figure 9 For the AVPYPQ and GABA in Experiment 3 of this application A Conformation diagram of R and OX2R molecules docking; Figure 10 YPVEP and GABA in Experiment 3 of this application A Conformation diagram of R and OX2R molecules docking; Figure 11 For the HLPIP and GABA in Experiment 3 of this application A Conformation diagram of R and OX2R molecules docking; Figure 12 VVPPF and GABA in Experiment 3 of this application A Conformation diagram of R and OX2R molecules docking; Figure 13 For the FPPQ and GABA in Experiment 3 of this application A Conformation diagram of R and OX2R molecules docking; Note: Appendix Figure 1-7 Different lowercase letters in abcd indicate significant differences between groups. P <0.05). Detailed Implementation

[0021] The present application will now be described in further detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and not intended to limit it. Furthermore, it should be noted that, for ease of description, only the parts relevant to the invention are shown in the accompanying drawings.

[0022] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. This application will now be described in detail with reference to the accompanying drawings and embodiments.

[0023] The first objective of this application is to provide a bovine colostrum active polypeptide with antioxidant and sleep-aiding effects, wherein the bovine colostrum active polypeptide has no obvious bitter or astringent taste.

[0024] The second objective of this application is to provide a method for preparing bovine colostrum active peptides with antioxidant and sleep-aiding effects. The preparation method has low equipment requirements, simple and easy-to-operate process, and is convenient for large-scale industrial production. The desired bovine colostrum active peptides can be obtained by simply combining operations such as mixing, pretreatment, enzymatic hydrolysis and filtration.

[0025] The third objective of this application is to propose the application of a bovine colostrum active polypeptide as described above, or a bovine colostrum active polypeptide prepared by the method described above, in the preparation of functional products with antioxidant and sleep-aiding effects.

[0026] To achieve the above objectives, this application provides a bovine colostrum bioactive polypeptide with antioxidant and sleep-aiding effects: The bovine colostrum active polypeptide contains at least one of HQPHQPLPPT, AVPYPQ, HLPIP, VVPPF, FPPQ, or YPVEP.

[0027] This application also provides a method for preparing bovine colostrum bioactive peptides with antioxidant and sleep-aiding effects, specifically including the following steps: (1) Mix bovine colostrum protein powder with water evenly, wherein the mass ratio of bovine colostrum protein powder to pure water is 1: (8-12), and shear at 20℃-30℃ for 10-30 min to obtain the first pretreatment solution.

[0028] (2) Add lipase (enzyme activity 10,000 U / g) to the first pretreatment solution, adjust the pH of the system to 6.5-7.5, hydrolyze at 35℃-39℃ for 0.5-1.5h, and then inactivate the enzyme at 85℃-90℃ for 15-20min to obtain the second pretreatment solution; wherein, the amount of lipase added is 4.0%-6.0% of the mass of bovine colostrum protein powder; (3) Add Bacillus licheniformis alkaline protease (enzyme activity 400,000 U / g) to the second pretreatment solution, adjust the pH of the system to 8.0-9.0, hydrolyze at 50℃-55℃ for 1.5-2.5 h, and then inactivate the enzyme at 85℃-90℃ for 15-20 min to obtain the first hydrolysate; wherein, the amount of Bacillus licheniformis alkaline protease added is 0.4%-0.6% of the mass of bovine colostrum protein powder; (4) Add bromelain (enzyme activity 200,000 U / g) to the first hydrolysate, adjust the pH of the system to 6.5-7.0, hydrolyze at 40℃-50℃ for 1.5-2.5h, and then inactivate the enzyme at 85℃-90℃ for 15-20min to obtain the second hydrolysate; wherein, the amount of bromelain added is 0.8%-1.2% of the mass of bovine colostrum protein powder.

[0029] (5) The second hydrolysate is cooled to room temperature and filtered. After filtration, the filtrate with a molecular weight of less than 5000 Da is obtained by ultrafiltration and then spray-dried to obtain the bovine colostrum active polypeptide.

[0030] (6) Activity detection and cell experiments were conducted on bovine colostrum active peptides, which showed that they inhibited ABTS, promoted the expression of the inhibitory neurotransmitter GABA and its receptor, and 5-hydroxytryptamine synaptic signals, inhibited neuronal excitability, regulated the sleep-wake cycle, and improved insomnia.

[0031] To verify the effectiveness of this application, the following embodiments and comparative examples are provided: It should be noted that: The enzymes with the same name used in the following examples and comparative examples are from the same commercially available company and brand, meaning that the enzyme activities of the enzymes with the same name used in each example and comparative example are consistent. The enzyme activities of the following enzymes used in the examples and comparative examples are as follows: Bacillus licheniformis alkaline protease 400,000 U / g, bromelain 200,000 U / g, pepsin 80,000 U / g, trypsin 250,000 U / g, papain 200,000 U / g, Bacillus subtilis neutral protease 500,000 U / g, flavor protease 100,000 U / g, acidic protease 600,000 U / g, proline endonuclease 150,000 U / g, and compound protease 100,000 U / g.

[0032] Example 1 1. Take 100g of bovine colostrum protein powder (protein content 50%), mix it evenly with 1000g of pure water, and shear at 25℃ for 20min; 2. Heat to 37℃, adjust pH to 7.0 with sodium hydroxide, add 5g of lipase for 1 hour of hydrolysis, and then heat to 85℃ for 20 minutes to inactivate the enzyme. 3. Cool to 55℃, adjust pH to 8.5 with sodium hydroxide, add 0.5g of Bacillus licheniformis alkaline protease to hydrolyze for 2h, and heat to 85℃ to inactivate enzyme for 20min; 4. After cooling to 45℃, adjust the pH to 6.8 with sodium hydroxide, add 1g of bromelain to hydrolyze for 2h, and heat to 85℃ to inactivate the enzyme for 20min; 5. After filtration and ultrafiltration, a bovine colostrum active polypeptide solution with a molecular weight of less than 5000 Da is obtained. 6. Obtain bovine colostrum active polypeptide powder by spray drying.

[0033] Example 2 1. Take 100g of bovine colostrum protein powder (protein content 50%), mix it evenly with 800g of pure water, and shear at 20℃ for 30min; 2. Heat to 39℃, adjust pH to 7.5 with sodium hydroxide, add 4g of lipase for hydrolysis for 1.5h, and then heat to 85℃ to inactivate the enzyme for 20min. 3. Cool to 50℃, adjust pH to 8.0 with sodium hydroxide, add 0.4g of Bacillus licheniformis alkaline protease to hydrolyze for 2.5h, and heat to 85℃ to inactivate enzyme for 20min; 4. After cooling to 40℃, adjust the pH to 6.5 with sodium hydroxide, add 1.2g of bromelain to hydrolyze for 1.5h, and heat to 90℃ to inactivate the enzyme for 20min; 5. After filtration and ultrafiltration, a bovine colostrum active polypeptide solution with a molecular weight of less than 5000 Da is obtained. 6. Obtain bovine colostrum active polypeptide powder by spray drying.

[0034] Example 3 1. Take 100g of bovine colostrum protein powder (protein content 50%), mix it evenly with 1200g of pure water, and shear at 30℃ for 10min; 2. Heat to 35℃, adjust pH to 6.5 with sodium hydroxide, add 6g of lipase to hydrolyze for 0.5h, and heat to 85℃ to inactivate enzyme for 20min; 3. Cool to 55℃, adjust pH to 9.0 with sodium hydroxide, add 0.6g of Bacillus licheniformis alkaline protease to hydrolyze for 1.5h, and heat to 90℃ to inactivate enzyme for 20min; 4. After cooling to 50℃, adjust the pH to 7.0 with sodium hydroxide, add 0.8g of bromelain and hydrolyze for 2.5h, then heat to 85℃ to inactivate the enzyme for 20min; 5. After filtration and ultrafiltration, a bovine colostrum active polypeptide solution with a molecular weight of less than 5000 Da is obtained. 6. Obtain bovine colostrum active polypeptide powder by spray drying.

[0035] Comparative Example 1 The hydrolysis process in this comparative example uses only Bacillus licheniformis alkaline protease. The specific experimental steps differ from those in Example 1 only in that the amount of Bacillus licheniformis alkaline protease added in step 3 is changed to 1g, and step 4 is removed. The operation and process of the remaining experimental steps are the same as those in Example 1.

[0036] In Example 1, the amount of Bacillus licheniformis alkaline protease added was 0.5g, and the amount of bromelain added was 1g. Based on the protein content (50%) of bovine colostrum protein powder, the total amount of enzyme added was 8000 U / g. The specific calculation process is as follows: The enzyme activity of 0.5g of Bacillus licheniformis alkaline protease is 400,000 U / g. 0.5g = 200,000 U, which translates to the enzyme addition per gram of bovine colostrum protein powder: 200,000 U / (100g) 50%)g=4000U / g (the denominator g in the unit represents the protein content per gram of bovine colostrum protein powder); similarly, the amount of bromelain added is 1g, which, based on the protein content of bovine colostrum protein powder, is 4000U / g.

[0037] Therefore, in this comparative example, the amount of Bacillus licheniformis alkaline protease added is 1g, which, based on the protein content in bovine colostrum protein powder, is 8000 U / g of Bacillus licheniformis alkaline protease added; this is the same as the total amount of Bacillus licheniformis alkaline protease and bromelain used in Example 1.

[0038] Comparative Example 2 The hydrolysis process of this comparative example uses Bacillus licheniformis alkaline protease and Bacillus subtilis neutral protease. The only difference between the specific experimental steps and those of Example 1 is that the bromelain in step 4 is replaced with Bacillus subtilis neutral protease, the amount added is 0.4g, the pH is 7.0, and the temperature is 55℃. The operation and process of the remaining experimental steps are the same as those of Example 1.

[0039] In this comparative example, the enzyme activity of 0.5g of Bacillus licheniformis alkaline protease was 400,000 U / g. 0.5g = 200,000 U, which translates to the enzyme addition per gram of bovine colostrum protein powder: 200,000 U / (100g) 50%)g=4000U / g (the denominator g in the unit represents the protein content per gram of bovine colostrum protein powder); similarly, the amount of Bacillus subtilis neutral protease added is 0.4g, and based on the protein content in bovine colostrum protein powder, the amount of Bacillus subtilis neutral protease added is 4000U / g; which is the same as the total amount of Bacillus licheniformis alkaline protease and bromelain used in Example 1.

[0040] Comparative Example 3 The hydrolysis process of this comparative example uses Bacillus licheniformis alkaline protease and flavor protease. The only difference between the specific experimental steps and those of Example 1 is that the bromelain in step 4 is replaced with flavor protease, the amount added is 2g, the pH is 7.0, and the temperature is 55℃. The operation and process of the remaining experimental steps are the same as those of Example 1.

[0041] In this comparative example, the enzyme activity of 0.5g of Bacillus licheniformis alkaline protease was 400,000 U / g. 0.5g = 200,000 U, which translates to the enzyme addition per gram of bovine colostrum protein powder: 200,000 U / (100g) 50%)g = 4000U / g (the denominator g in the unit represents the protein content per gram of bovine colostrum protein powder); similarly, the amount of flavor protease added is 2g, and based on the protein content in bovine colostrum protein powder, the amount of flavor protease added is 4000U / g; which is the same as the total amount of Bacillus licheniformis alkaline protease and bromelain used in Example 1.

[0042] Comparative Example 4 The hydrolysis process of this comparative example uses Bacillus licheniformis alkaline protease and pepsin. The only difference between the specific experimental steps and those of Example 1 is that the bromelain in step 4 is replaced with pepsin, the amount added is 2.5g, the pH is 3, and the temperature is 37℃. The operation and process of the remaining experimental steps are the same as those of Example 1.

[0043] In this comparative example, the enzyme activity of 0.5g of Bacillus licheniformis alkaline protease was 400,000 U / g. 0.5g = 200,000 U, which translates to the enzyme addition amount per gram of bovine colostrum protein powder: 200,000 U / (100g) 50%)g = 4000U / g (the denominator g in the unit represents the protein content per gram of bovine colostrum protein powder); similarly, the amount of pepsin added is 2.5g, which, based on the protein content of bovine colostrum protein powder, is 4000U / g of pepsin enzyme added; this is the same as the total amount of Bacillus licheniformis alkaline protease and bromelain used in Example 1.

[0044] Comparative Example 5 The hydrolysis process of this comparative example uses Bacillus licheniformis alkaline protease and trypsin. The only difference between the specific experimental steps and those of Example 1 is that the bromelain in step 4 is replaced with trypsin, the amount added is 0.8g, the pH is 7.5, and the temperature is 50℃. The operation and process of the remaining experimental steps are the same as those of Example 1.

[0045] In this comparative example, the enzyme activity of 0.5g of Bacillus licheniformis alkaline protease was 400,000 U / g. 0.5g = 200,000 U, which translates to the enzyme addition amount per gram of bovine colostrum protein powder: 200,000 U / (100g) 50%)g = 4000U / g (the denominator g in the unit represents the protein content per gram of bovine colostrum protein powder); similarly, the amount of trypsin added is 0.8g, which, based on the protein content of bovine colostrum protein powder, is 4000U / g of trypsin enzyme added; this is the same as the total amount of Bacillus licheniformis alkaline protease and bromelain used in Example 1.

[0046] Comparative Example 6 The hydrolysis process of this comparative example uses Bacillus licheniformis alkaline protease and papain. The only difference between the specific experimental steps and those of Example 1 is that the bromelain in step 4 is replaced with papain, the amount added is 1g, the pH is 6.5, and the temperature is 55℃. The operation and process of the remaining experimental steps are the same as those of Example 1.

[0047] In this comparative example, the enzyme activity of 0.5g of Bacillus licheniformis alkaline protease was 400,000 U / g. 0.5g = 200,000 U, which translates to the enzyme addition amount per gram of bovine colostrum protein powder: 200,000 U / (100g) 50%)g = 4000U / g (the denominator g in the unit represents the protein content per gram of bovine colostrum protein powder); similarly, the amount of papain added is 1g, which, based on the protein content of bovine colostrum protein powder, is 4000U / g; the same amount as the total amount of Bacillus licheniformis alkaline protease and bromelain used in Example 1.

[0048] Comparative Example 7 The hydrolysis process of this comparative example uses Bacillus licheniformis alkaline protease and acidic protease. The specific experimental steps are different from those in Example 1 only in that the amount of Bacillus licheniformis alkaline protease added in step 3 is changed to 0.4g, the bromelain in step 4 is changed to acidic protease, the amount added is 0.4g, the pH is 3, and the temperature is 37℃. The operation and process of the remaining experimental steps are the same as those in Example 1.

[0049] In this comparative example, the enzyme activity of 0.4 g of Bacillus licheniformis alkaline protease was 400,000 U / g. 0.4g = 160,000 U, which translates to the enzyme addition per gram of bovine colostrum protein powder: 160,000 U / (100g) 50%)g=3200U / g (the denominator g in the unit represents the protein content per gram of bovine colostrum protein powder); similarly, the amount of acidic protease added is 0.4g, and based on the protein content in bovine colostrum protein powder, the amount of acidic protease added is 4800U / g; which is the same as the total amount of Bacillus licheniformis alkaline protease and bromelain used in Example 1.

[0050] Comparative Example 8 The hydrolysis process in this comparative example uses Bacillus licheniformis alkaline protease and proline endopeptidase. The specific experimental steps differ from those in Example 1 only in that the amount of Bacillus licheniformis alkaline protease added in step 3 is changed to 0.475g, and the bromelain in step 4 is replaced with proline endopeptidase with an added amount of 1.4g. The pH is 5.0 and the temperature is 60℃. The operation and process of the remaining experimental steps are the same as those in Example 1.

[0051] In this comparative example, the enzyme activity of 0.475 g of Bacillus licheniformis alkaline protease was 400,000 U / g. 0.475g = 190,000 U, which translates to the enzyme addition amount per gram of bovine colostrum protein powder: 190,000 U / (100g) 50%)g=3800U / g (the denominator g in the unit represents the protein content per gram of bovine colostrum protein powder); similarly, the amount of proline endonuclease added is 1.4g, which, based on the protein content in bovine colostrum protein powder, is 4200U / g of proline endonuclease added; this is the same as the total amount of Bacillus licheniformis alkaline protease and bromelain used in Example 1.

[0052] Comparative Example 9 The hydrolysis process of this comparative example uses Bacillus licheniformis alkaline protease and a compound protease. The only difference between the specific experimental steps and those of Example 1 is that the bromelain in step 4 is replaced with a compound protease, the amount added is 2g, the pH is 7.0, and the temperature is 55℃. The operation and process of the remaining experimental steps are the same as those of Example 1.

[0053] In this comparative example, the enzyme activity of 0.5g of Bacillus licheniformis alkaline protease was 400,000 U / g. 0.5g = 200,000 U, which translates to the enzyme addition per gram of bovine colostrum protein powder: 200,000 U / (100g) 50%)g = 4000U / g (the denominator g in the unit represents the protein content per gram of bovine colostrum protein powder); similarly, the amount of compound protease added is 2g, and based on the protein content in bovine colostrum protein powder, the amount of compound protease added is 4000U / g; which is the same as the total amount of Bacillus licheniformis alkaline protease and bromelain used in Example 1.

[0054] The yields of bovine colostrum bioactive peptides prepared in Examples 1-3 and Comparative Examples 1-9 of this application are shown in Table 1. Table 1. Yields of bovine colostrum bioactive peptides prepared in Experiments 1-3 and Comparative Examples 1-9

[0055] Table 1 shows that the yield of Comparative Example 1 was lower, while the yields of the other examples and comparative examples were higher. Comparative Example 1 was hydrolyzed with a single enzyme, indicating that the effect of dual-enzyme hydrolysis is better than that of single-enzyme hydrolysis. At the same time, the yields of each group were basically stable at around 60%. The peptides in bovine colostrum were obtained after effective enzymatic hydrolysis, which is more conducive to further screening of active peptides.

[0056] This application also provides the following experiments for activity detection and peptide screening of the bovine colostrum bioactive peptides obtained in this application: Experiment 1: Determination of ABTS inhibition rate The bovine colostrum active peptides obtained in Examples 1-3 and Comparative Examples 1-9 were prepared into solutions of 20 mg / mL, and the ABTS inhibition rate was determined. The detection method was in accordance with GB / T 39100-2020, ABTS method for peptide antioxidant determination. The determination data are summarized in Table 2. Table 2. ABTS inhibition rates of bovine colostrum bioactive peptides obtained in Examples 1-3 and Comparative Examples 1-9

[0057] According to the data in Table 2, the bovine colostrum active peptides prepared in Examples 1-3 and Comparative Examples 1-9 all have a certain ABTS inhibition ability. However, the inhibition effect of the examples is better than that of the comparative examples. Among them, the bovine colostrum active peptides prepared in Example 1 have the highest ABTS inhibition rate of 72.98%, indicating that the bovine colostrum active peptides have good antioxidant capacity.

[0058] Experiment 2: Sleep-aiding effect of bovine colostrum bioactive peptides In this experiment, a neural cell line PC12 cell differentiation model was established for the control group (CON group) and experimental group. Based on the comprehensive in vitro antioxidant index, Example 1 and comparative examples 2, 6, 7 and 9 were selected for cell experiments. The PC12 cell differentiation model was established by adding complete culture medium containing NGF to the culture flasks, with a final NGF concentration typically of 50 ng / mL. The culture flasks were then placed back in an incubator and cultured at 37°C and 5% CO2. During the differentiation induction process, the NGF-containing complete culture medium was replaced every 1–2 days. The cells were then induced to differentiate using NGF until the appropriate time was reached.

[0059] 2.1 Determination of the optimal experimental concentration of bovine colostrum bioactive peptides Cell viability was determined using the CCK8 (Cell Counting Kit-8) method. After significance analysis, a concentration of 1.2 mg / mL, which is non-toxic to cells, was selected as the cell drug concentration.

[0060] 2.2 Effects of bovine colostrum bioactive peptides on GABA content in PC12 cells Cell culture: PC12 cells were seeded in DMEM medium containing 10% fetal bovine serum and cultured in a cell culture incubator at 37°C and 5% CO2. Experiments were conducted when the cells reached the logarithmic growth phase.

[0061] Induction of differentiation: NGF was added to the control group and each experimental group to achieve a suitable induction concentration (usually 50-100 ng / mL), and the cells were cultured for a certain period of time (usually 3-7 days) to induce PC12 cell differentiation.

[0062] Cell collection and lysis: After culture, discard the culture medium and gently wash the cells 2-3 times with PBS. Add an appropriate amount of cell lysis buffer and incubate on ice for 15-30 min to allow for complete cell lysis. Then transfer the cell lysate to centrifuge tubes and centrifuge at 10,000-12,000 rpm for 10-15 min at 4°C. Use the supernatant for ELISA detection.

[0063] GABA content detection: Follow the instructions for the GABA ELISA detection kit.

[0064] Gamma-aminobutyric acid (GABA) is a major inhibitory neurotransmitter in the brain and is crucial for the central nervous system. GABA primarily plays a sleep-inducing role in the ventrolateral preoptic nucleus of the hypothalamus. It inhibits neuronal excitability by binding to specific receptors and participates in regulating the sleep-wake cycle. GABA has wide applications in clinical research, including the treatment of insomnia, hypertension, stress, and increasing serum growth hormone levels.

[0065] Figure 1 The graph shows the data analysis of intracellular γ-aminobutyric acid (GABA) content in PC12 cells. Detailed data are shown in Table 3. Table 3. Effects of bovine colostrum bioactive peptides on intracellular γ-aminobutyric acid (GABA) content in PC12 cells.

[0066] according to Figure 1 It can be seen that, compared with the control group, bovine colostrum active peptides increased the content of γ-aminobutyric acid in PC12 cells. The improvement effect was significant in Example 1 and Comparative Examples 2, 6 and 7, especially in Example 1, which increased the content to 2.5 times that of the control group. This indicates that bovine colostrum active peptides further inhibited neuronal excitability by enhancing the secretion of γ-aminobutyric acid in cells.

[0067] 2.3 Effects of bovine colostrum bioactive peptides on the expression levels of key genes in PC12 cells Cell culture: PC12 cells were seeded into cell culture flasks, and DMEM medium containing 10% fetal bovine serum was added. The cells were cultured in a cell culture incubator at 37°C and 5% CO2. When the cells reached the logarithmic growth phase, PC12 cells were treated with NGF to induce differentiation.

[0068] Drug incubation: Aspirate the culture medium from the culture dish and gently wash the cells 2-3 times with PBS. Add fresh culture medium containing 1.2 mg / mL bovine colostrum active peptides to the culture dishes of each experimental group and control group, and continue to culture in a cell culture incubator for 24-48 h to allow the drug to fully act on the cells.

[0069] Key gene detection: After drug incubation, RNA was extracted, reverse transcribed, and Real-time qPCR was performed to detect key sleep genes. Primers were designed and synthesized by Sangon Biotech (Shanghai) Co., Ltd., and the primer sequences are listed in Table 4 below; the β-actin gene was used as an internal reference gene, with 3 replicates in each group; the relative expression levels of each gene were calculated using 2-ΔΔCt.

[0070] Table 4 Primer sequence listing

[0071] The sleep-wake cycle is a complex physiological process regulated by various neurotransmitters and neural circuits, with neurons being the basic unit of these regulatory mechanisms. Neurons regulate their excitability through rhythmic changes in gene expression, such as GABA receptor genes, thereby controlling the timing of sleep and wakefulness.

[0072] When GABAergic neurons are active in the ventrolateral anterior hypothalamus, their expression level is higher than in the waking state, directly inhibiting the arousal center and thus promoting sleep. Figure 2 The effect of bovine colostrum bioactive peptides on GABA mRNA expression levels in PC12 cells is detailed in Table 5. Table 5. Effects of bovine colostrum bioactive peptides on GABA mRNA expression levels in PC12 cells.

[0073] according to Figure 2 It can be seen that, compared with the control group, Example 1 significantly increased the expression level of GABA gene, thereby affecting the central nervous system and promoting sleep.

[0074] OX1R (orexin type 1 receptor) binds to the wakefulness-promoting orexin neuropeptide, activating the brain's wakefulness system and inhibiting sleep. OX2R (orexin type 2 receptor) is involved in the central control of food intake, the maintenance of sleep, energy metabolism, and neuroendocrine homeostasis. Clinically used OX1R or OX2R antagonists induce sleep by blocking the OX1R or OX2R signaling pathways. Figure 3 The effect of bovine colostrum bioactive peptides on the expression level of OX1R mRNA in PC12 cells. Figure 4 The effect of bovine colostrum bioactive peptides on the expression level of OX2R mRNA in PC12 cells was investigated. Detailed data are shown in Tables 6-7. Table 6. Effects of bovine colostrum bioactive peptides on OX1R mRNA expression levels in PC12 cells.

[0075] Table 7. Effects of bovine colostrum bioactive peptides on OX2R mRNA expression levels in PC12 cells.

[0076] according to Figure 3 , 4 It can be seen that Example 1 and the comparative example can significantly reduce the expression level of the OX1R gene, and Example 1 and the comparative example 9 can reduce the expression level of the OX2R gene, inhibit the OX1R or OX2R signaling pathway, and induce sleep.

[0077] GABA AThe R gene is the gene encoding the GABA class A receptor. A R is a pentamer chloride ion channel composed of subunits such as α, β, and γ. After activation, chloride ion influx leads to neuronal hyperpolarization and reduces excitability. Figure 5 Bovine colostrum active peptides against intracellular GABA in PC12 cells A The effect of R mRNA expression levels is detailed in Table 8: Table 8. Effects of bovine colostrum bioactive peptides on GABAAR mRNA expression levels in PC12 cells.

[0078] according to Figure 5 It can be seen that Example 1 and the comparative example significantly improved the GABA gene. A The expression level of R, especially in Example 1, was 9.25 times that of the control group. GABA A The upregulation of R mRNA further confirms the promoting effect of bovine colostrum active peptides on GABA production, thereby exerting a sleep-aiding effect.

[0079] The 5-HT gene encodes the monoamine neurotransmitter serotonin. After binding to 5-HT receptors in the central and peripheral nervous systems, 5-HT plays an important role in regulating mood, sleep, and appetite. Figure 6 The effect of bovine colostrum bioactive peptides on the expression level of 5-HT mRNA in PC12 cells is detailed in Table 9. Table 9. Effects of bovine colostrum bioactive peptides on 5-HT mRNA expression levels in PC12 cells.

[0080] according to Figure 6 It can be seen that, compared with the control group, Example 1 and Comparative Examples 2, 6, 7 and 9 significantly enhanced the expression of 5-HT gene in cells, indicating that bovine colostrum active peptides exert a sleep-aiding effect by promoting 5-HT secretion and inhibiting neuronal excitability.

[0081] TNF-α is a pleiotropic pro-inflammatory cytokine. When produced in excess, it drives an abnormal immune response and continuously amplifies inflammatory signals. Figure 7 The effect of bovine colostrum peptide on TNF-α mRNA expression in PC12 cells is detailed in Table 10. Table 10 Effects of bovine colostrum bioactive peptides on TNF-α mRNA expression levels in PC12 cells

[0082] according to Figure 7It can be seen that, compared with the control group, the expression of TNF-α gene in cells of all groups was low after treatment with bovine colostrum active peptides, especially in Example 1 and Comparative Example 9, indicating that bovine colostrum active peptides have the effects of repairing cell damage, reducing inflammatory response, and indirectly improving insomnia.

[0083] The combined expression data of the six genes showed that the expression levels of GABA and its receptor genes were significantly increased after intervention with bovine colostrum bioactive peptides in Example 1. This indicates that bovine colostrum bioactive peptides regulate the sleep-wake cycle by promoting the expression of the inhibitory neurotransmitter GABA and its receptor, thereby inhibiting neuronal excitability. Simultaneously, the expression levels of OX1R and OX2R genes were significantly decreased, indicating that key upstream controllers of excitatory neurotransmitters were inhibited, thus improving insomnia. Furthermore, the 5-HT gene level was significantly increased, suggesting that bovine colostrum bioactive peptides mediate the 5-HT synaptic signaling pathway and contribute to improving insomnia. The expression level of the TNF-α gene was significantly reduced after intervention with bovine colostrum bioactive peptides, reducing inflammatory responses and promoting sleep.

[0084] Experiment 3: Peptide screening of bioactive polypeptides from bovine colostrum In this experiment, the active peptides of bovine colostrum prepared in Example 1 were screened and analyzed.

[0085] 3.1 Pretreatment Method 3.1.1 Sample Dissolution 1) Accurately weigh 1 mg of sample into a centrifuge tube, add 100 μL of PBS, and vortex to mix. 3.1.2 Reductive alkylation 1) Accurately pipette 1 μL of 1M DTT solution into the sample to make the final DTT concentration 10 mmol / L, and reduce it in a 56℃ water bath for 1 h; 2) Accurately pipette 2 μL of 1M IAM solution into the sample to make the final IAM concentration 20 mmol / L, and react in the dark at room temperature for 40 min. 3) Accurately pipette 1 μL of 1M DTT solution into the sample to make the final DTT concentration 10 mmol / L, in order to neutralize unreacted IAM.

[0086] 3.1.3 Desalination Desalting was performed using C18 stage-tip, followed by vacuum drying at 45°C.

[0087] 3.2 Computer Usage Requirements 3.2.1 Liquid Chromatography Conditions 1) Pre-column: 150 μm id × 50 mm, packing: Reprosil-Pur 120 C18-AQ 3 μm; Analytical column: 150 μm id × 170 mm, packing: Reprosil-Pur 120 C18-AQ 1.9 μm 2) Mobile phase A: 0.1% FA; 3) Mobile phase B: 0.1% FA, 80% ACN; 4) Flow rate: 600 nL / min; 5) Analysis time for each component: 66 min; 6) Specific chromatographic conditions are shown in Table 11: Table 11 Chromatographic conditions

[0088] 3.2.2 Mass Spectrometry Conditions Acquisition mode: DAA Level 1 mass spectrometry parameters: 1) Resolution: 70,000 2) AGCtarget: 3e6 3) Maximum IT: 100ms 4) Scanrange: 100~1500m / z Secondary mass spectrometry parameters: 1) Resolution: 17,500 2) AGCtarget: 1e5 3) Maximum IT: 50ms 4) TopN: 20 5) NCE / Stepped NCE: 28 Raw data was obtained through mass spectrometry.

[0089] 3.3 Search Criteria The raw mass spectrometry files were searched using software to retrieve the target protein database. The search parameters were as follows: 1) Fixed modifications: Carbamidomethyl (C).

[0090] 2) Variable modifications: Oxidation (M), Acetyl (Peptide N-term).

[0091] 3) Enzyme: Non specific.

[0092] 4) Database: uniprotkb_proteome_UP000009136_2025_12_17.fasta.

[0093] 5) Peptide Mass Tolerance: 20 ppm 6) Secondary mass spectrometry bias (Fragment Mass Tolerance): 0.02 Da 3.4 Peptide Screening and Molecular Docking Raw files acquired by mass spectrometry were searched for peptides with an abundance greater than 1,000,000,000. After molecular docking, 20 peptides were identified, and their activities were predicted using our company's proprietary active peptide prediction website (http: / / 156.232.99.201:9090 / ). Peptide-related information is listed in Table 12. Furthermore, the following 20 peptides were compared with GABA... A Molecular docking was performed on the R (PDB: 6HUP) and OX2R (PDB: 4S0V) receptors, and the results are summarized in Table 12.

[0094] Table 12 Peptide Information and Receptor Binding Energy

[0095] GABA A R (PDB ID: 6HUP) and OX2R (PDB ID: 4S0V) are two important targets related to sleep and key receptor proteins for improving sleep in clinical sleep aids, playing a crucial role in the treatment of insomnia.

[0096] GABA A R plays a particularly important role in mediating the sleep regulation function of GABA, and it is mainly found in the hippocampus, hypothalamus, and cerebral cortex. A Allosteric sites on R are also molecular targets for anti-anxiety and hypnotic drugs.

[0097] OX2R (orexin receptor-2) dominates the process of wakefulness and non-rapid eye movement sleep, and has unique therapeutic effects in the field of insomnia treatment, gradually becoming a new focus of insomnia drug research and development.

[0098] Molecular docking software was used to dock the 20 selected fragments with the two targets mentioned above. The binding energies of the peptides to the receptors are summarized in Table 12; among them, GABA A The 3D structural diagrams of R (PDB: 6HUP) and OX2R (PDB: 4S0V) can be downloaded from the PDB database (http: / / www.rcsb.org / ).

[0099] As shown in Table 12, all 20 peptides are associated with GABA. A R and OX2R have a certain binding energy. After the peptide binds to the receptor, it has an effect. It has potential therapeutic significance for insomnia by promoting both inhibitory neurotransmitter pathways and inhibiting excitatory neurotransmitter pathways.

[0100] Based on binding energy and peptide abundance, several sequences, including HQPHQPLPPT, AVPYPQ, YPVEP, HLPIP, VVPPF, and FPPQ, were screened as characteristic peptide sequences in bovine colostrum active polypeptides that have significant sleep-promoting effects.

[0101] like Figure 8 As shown, HQPHQPLPPT and GABA A The interactions between R are hydrophobic and hydrogen-bonded, and the interactions with OX2R are hydrophobic, hydrogen-bonded, and salt-bridged; for example Figure 9 As shown, AVPYPQ and GABA A The interactions between R are hydrophobic and hydrogen-bonded, and the interactions with OX2R are hydrophobic, hydrogen-bonded, and salt-bridged; for example Figure 10 As shown, YPVEP and GABA A The interactions between R and OX2R are hydrophobic, hydrogen-bonded, and salt-bridged; the interactions with OX2R are also hydrophobic, hydrogen-bonded, and salt-bridged. Figure 11 As shown, HLPIP and GABA A The interactions between R are hydrophobic and hydrogen-bonded, and the interactions with OX2R are hydrophobic, hydrogen-bonded, and salt-bridged; for example Figure 12 As shown, VVPPF and GABA A The interactions between R are hydrophobic, hydrogen-bonded, and salt-bridged; the interactions with OX2R are hydrophobic and hydrogen-bonded. Figure 13 As shown, FPPQ and GABA A The R groups interact with each other through hydrophobic interactions, hydrogen bonds, and salt bridges, and with OX2R through hydrophobic interactions, hydrogen bonds, and π-π bonds.

[0102] In addition, flavor testing showed that the bovine colostrum active peptides prepared in the embodiments of this application had no obvious bitterness or astringency.

[0103] In summary, the proposed solution has the following advantages: The bovine colostrum active polypeptide prepared by the method of this application contains at least one of HQPHQPLPPT, AVPYPQ, HLPIP, YPVEP, VVPPF, or FPPQ. The prepared bovine colostrum active polypeptide has important biological activities: on the one hand, it exerts an antioxidant effect by scavenging / inhibiting ABTS free radicals; on the other hand, it inhibits neuronal excitability, regulates the sleep-wake cycle, and improves insomnia by promoting the expression of the inhibitory neurotransmitter GABA and its receptor, as well as 5-hydroxytryptamine synaptic signals.

[0104] Furthermore, the bovine colostrum active peptides prepared by the method of this application have no obvious bitter or astringent taste. Moreover, the preparation method of this application has low equipment requirements, is simple and easy to operate, and can obtain the desired bovine colostrum active peptides through a simple combination of operations such as mixing, pretreatment, enzymatic hydrolysis, and filtration, facilitating large-scale industrial production.

[0105] It should be noted that: (1) Definition: In this article, “~” is used to represent the range of values, and the range of values ​​represented by this expression includes two endpoint values.

[0106] The term "Da" used in this article stands for Dalton, a commonly used unit for molecular weight.

[0107] The terms "ultrafiltration," "filtration," and "spray drying" used in this article are conventional names for processing steps in the field, and their names accurately describe the processing procedures, so they will not be repeated here.

[0108] (2) Raw materials used in implementation: All enzymes used in this article are commercially available enzymes that can be purchased and obtained by those skilled in the art.

[0109] In summary, the specific parameters or some commonly used reagents or raw materials in the above embodiments are specific or preferred embodiments under the concept of this application, and not limitations thereof; those skilled in the art can make adaptive adjustments within the concept and protection scope of this application.

[0110] In addition, unless otherwise specified, the raw materials used may be commercially available products in the field or prepared by conventional methods in the field; that is, the reagents and instruments used in this embodiment do not specify the manufacturer or other information, and are all conventional products that can be purchased from the market.

[0111] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

[0112] The specific embodiments of this application have been described above, but the scope of protection of this application is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the scope of the technology disclosed in this application should be included within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A method for preparing bovine colostrum bioactive polypeptides, characterized in that, Includes the following steps: S1. Mix bovine colostrum protein powder with water evenly and shear it to obtain a pretreatment solution. S2. Add lipase to the primary pretreatment solution for secondary pretreatment, and then inactivate the enzyme to obtain a secondary pretreatment mixture. S3. Add Bacillus licheniformis alkaline protease to the secondary pretreatment solution for the first hydrolysis, and then inactivate the enzyme to obtain the first hydrolysate; S4. Add bromelain to the first hydrolysate for a second hydrolysis, and then inactivate the enzyme to obtain the second hydrolysate; S5. The second hydrolysate is subjected to filtration, ultrafiltration and spray drying to obtain powdered bovine colostrum active peptides.

2. The preparation method according to claim 1, characterized in that, In step S1, the mass ratio of bovine colostrum protein powder to water is 1:(8~12); the shearing treatment lasts for 10~30 min and the temperature is 20℃~30℃. In step S2, the amount of lipase added is 4.0% to 6.0% of the mass of the bovine colostrum protein powder; the temperature of the secondary pretreatment is 35℃ to 39℃, the reaction pH is 6.5 to 7.5, and the time is 0.5 to 1.5 h.

3. The preparation method according to claim 1, characterized in that, In step S3, the amount of Bacillus licheniformis alkaline protease added is 0.4% to 0.6% of the mass of the bovine colostrum protein powder. In step S4, the amount of bromelain added is 0.8% to 1.2% of the mass of the bovine colostrum protein powder.

4. The preparation method according to claim 1, characterized in that, In step S3, the temperature of the first hydrolysis is 50℃~55℃, the reaction pH is 8.0~9.0, and the hydrolysis time is 1.5~2.5h; In step S4, the temperature of the second hydrolysis is 40℃~50℃, the reaction pH is 6.5~7.0, and the hydrolysis time is 1.5~2.5h.

5. The preparation method according to claim 1, characterized in that, In S2, S3 and S4, the enzyme inactivation conditions are all at 85℃~90℃ for 15~20min. In step S5, the ultrafiltration process is performed using an ultrafiltration membrane with a molecular weight cutoff of 5000 Da.

6. A bovine colostrum bioactive polypeptide, characterized in that, It is prepared by the preparation method described in any one of claims 1-5.

7. The bovine colostrum active polypeptide according to claim 6, characterized in that, The bovine colostrum active polypeptide contains at least one of the peptide sequences HQPHQPLPPT, AVPYPQ, HLPIP, VVPPF, YPVEP, or FPPQ.

8. A functional product, characterized in that, Its active component comprises bovine colostrum active polypeptides as described in any one of claims 6-7.

9. The use of a bovine colostrum active polypeptide as described in any one of claims 6-7 in the preparation of functional products with antioxidant and / or sleep-aiding effects.

Images