Probiotic-carrying modified milk-derived active peptide and application thereof in sleep-aiding milk beverage

Abstract

The invention provides a modified milk-derived active peptide loaded with probiotics and application of the modified milk-derived active peptide in a sleep-aiding milk beverage, polyethylene glycol is introduced into casein hydrolysate decapeptide YLGYLEQLLR for modification to prepare modified polypeptide, hydrogel is formed based on self-assembly of the modified polypeptide, and the probiotics are embedded to prepare the modified milk-derived active peptide loaded with the probiotics. The modified milk-derived active peptide can embed and slowly release the probiotics, so that the probiotics are continuously released in vivo, and the sleep quality is improved; in addition, the probiotics and the polypeptide synergistically play a role, so that the mood is regulated, and the sleep quality is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of dairy processing, and specifically relates to a modified milk-derived active peptide carrying probiotics and its application in a sleep-aiding milk beverage. Background Art

[0002] With the accelerating pace of life and increasing work pressure, more and more people are facing sleep problems. According to relevant statistics, approximately one-third of people worldwide suffer from sleep problems, with insufficient sleep being a prominent issue. Sleep problems not only affect people's daily lives and work efficiency, but can also lead to other issues such as anxiety and depression. Currently, the sleep-aiding products on the market are basically pharmaceutical products, mainly antidepressants and melatonin drugs. Long-term use will not only lead to drug dependence, but may also cause serious sleep disorders; and some health care products have limited effects and cannot bring any benefits to the patient's body. Existing studies have shown that probiotics can regulate the levels of host neurotransmitters (such as 5-hydroxytryptamine) through the gut-brain axis, thereby improving sleep disorders and anxiety. However, probiotics are easily destroyed by gastric acid, bile salts and digestive enzymes during oral delivery, resulting in a significant decrease in the number of live bacteria reaching the intestine (survival rate <30%), which seriously limits its actual application effect. In addition, free probiotics have a short colonization time in the intestine and cannot continuously release active ingredients to achieve long-term regulation. There is an urgent need to develop a new protective delivery system. The sleep-inducing decapeptide YLGYLEQLLR has been identified in casein enzymatic hydrolysis products. It can cross the blood-brain barrier and bind to neuronal serotonin receptors, directly regulating the sleep-wake cycle. However, natural casein peptides are rapidly degraded into small molecular fragments by pepsin in the stomach, resulting in loss of activity. Therefore, it is necessary to develop a system that encapsulates probiotics with modified peptides for application in sleep-inducing foods. Summary of the Invention

[0003] Technical Problem to be Solved: In response to the above technical problems, the present invention aims to provide a modified milk-derived active peptide carrying probiotics and its use in a sleep-aiding milk beverage. The modified peptide is prepared by introducing polyethylene glycol into the casein hydrolysate decapeptide YLGYLEQLLR. The modified milk-derived active peptide self-assembles into a hydrogel, which then encapsulates probiotics to produce a modified milk-derived active peptide carrying probiotics. The modified milk-derived active peptide encapsulates and slowly releases the probiotics, allowing them to be continuously released in the body, improving sleep quality. Furthermore, the probiotics and peptide work synergistically to regulate mood and improve sleep quality.

[0004] Technical solution: A modified milk-derived active peptide carrying probiotics, wherein the modified milk-derived active peptide is self-assembled to form a hydrogel and the probiotics are embedded in the hydrogel; The modified milk-derived active peptide is obtained by introducing polyethylene glycol into the casein hydrolysate decapeptide YLGYLEQLLR. The preparation method of the modified milk-derived active peptide carrying probiotics is as follows: S1. Dissolve the casein hydrolysate decapeptide in PBS buffer, add polyethylene glycol, stir at room temperature for 10-12 hours, dialyze, and lyophilize to obtain a modified polypeptide powder; S2. The modified polypeptide powder was dissolved in a CaCl2 solution at a concentration of 2-5% to obtain a modified polypeptide solution; S3. Cultivate the probiotics to the logarithmic phase, collect the cells by centrifugation, wash with sterile PBS buffer, and resuspend to prepare a probiotic suspension; S4. Mix the modified polypeptide solution and the probiotic suspension, adjust the pH to 6.5-7.5, let stand at 30-37°C for 2-5 hours, and freeze-dry to obtain the modified milk-derived active peptide carrying the probiotics. Furthermore, the concentration of casein hydrolysate decapeptides in step S1 is 10-15 mg / mL; and the mass ratio of polyethylene glycol to casein hydrolysate decapeptides is (1-3):1. Furthermore, the concentration of the modified polypeptide solution in step S2 is 2-10 mg / mL. Furthermore, the probiotics in step S3 are any one of Lactobacillus plantarum, Bifidobacterium, and Lactobacillus fermentum; the concentration of the probiotic suspension is 1×10 8 -1×10 9 CFU / mL. Furthermore, in step S4, the volume ratio of the modified polypeptide solution to the probiotic suspension is (1-2.5):1. Application of the modified milk-derived active peptide carrying probiotics prepared by the above preparation method in a sleep-aiding milk beverage. Furthermore, the formula of the sleep-aiding milk beverage is as follows: by mass percentage, 65-70% raw cow's milk; 1-2% light cream; 0.05-0.2% γ-aminobutyric acid; 0.05-0.2% jujube kernel powder; 0.02-1% yam powder; 0-0.005% edible salt; 0.1-0.5% emulsion stabilizer; 1-5% erythritol; 0-0.005% sucralose; 0-0.03% sodium carbonate; 0-0.07% edible flavoring; 2-5% modified milk-derived active peptides carrying probiotics. Beneficial effects: 1. The modified milk-derived active peptide prepared in the present invention is based on a decapeptide from casein hydrolysate. The decapeptide YLGYLEQLLR has a sleep-inducing function and can bind to neuronal serotonin receptors across the blood-brain barrier, directly regulating the sleep-wake cycle. However, natural casein peptides are rapidly degraded into small molecular fragments by pepsin in the gastric environment, resulting in loss of activity and an inability to effectively encapsulate probiotics and deliver them to the intestines. Therefore, the present invention grafts the decapeptide from casein hydrolysate with polyethylene glycol. The polyethylene glycol is covalently bound to the amino or carboxyl groups of the polypeptide, forming a steric hindrance that reduces the contact between gastric acid and digestive enzymes and the polypeptide, thereby increasing the stability of the polypeptide hydrogel in the stomach. 2. In order to further improve the stability of the polypeptide in the stomach, the present invention also adds Ca 2+ Calcium ions can bind to the carboxyl groups on the casein peptide chains through electrostatic interactions to form salt bridges, thereby enhancing the cross-linking between peptide chains and improving the stability of the polypeptide hydrogel; 3. The modified milk-derived active peptide prepared by the present invention also embeds probiotics. The porous structure of the polypeptide hydrogel has a sustained-release effect on the probiotics, so that the delivered probiotics can regulate the production of 5-hydroxytryptamine in the intestinal environment, and can enter the nerve cells with the blood, bind to the 5-hydroxytryptamine receptors between neurons, and play a role in regulating emotions, thereby playing a role in preventing sleep disorders, improving sleep quality, and alleviating anxiety. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 Graph showing the sleep time of insomniac mice in the control group, Example 10 group, and Comparative Examples 8-15 groups; Figure 2 Graph showing the sleep latency of insomniac mice in the control group, Example 10 group, and Comparative Examples 8-15 groups; Figure 3 5-HT content in brain tissue of insomnia mice in the control group, Example 10 group, and Comparative Examples 8-15 groups. DETAILED DESCRIPTION The present invention will be further described below with reference to the accompanying drawings and embodiments. The following embodiments are intended to explain the present invention, but the present invention is not limited to the following embodiments: Example 1 A modified milk-derived active peptide carrying probiotics, the specific preparation steps are as follows: S1. Dissolve the casein hydrolysate decapeptide in PBS buffer to a concentration of 10 mg / mL. Add polyethylene glycol at a mass ratio of 1:1. Stir at room temperature for 12 h, dialyze, and lyophilize to obtain a modified polypeptide powder. S2. The modified polypeptide powder was dissolved in a 3% CaCl2 solution to obtain a modified polypeptide solution having a concentration of 5 mg / mL; S3. Lactobacillus plantarum was cultured to the logarithmic phase, the cells were collected by centrifugation, washed with sterile PBS buffer, and resuspended to obtain a concentration of 1×10 9 CFU / mL of probiotic suspension; S4. The modified polypeptide solution and the probiotic suspension were mixed in a volume ratio of 2:1, the pH was adjusted to 7.0, the mixture was allowed to stand at 37°C for 4 hours, and freeze-dried to obtain the modified milk-derived active peptide carrying the probiotics. Example 2 A modified milk-derived active peptide carrying probiotics, the specific preparation steps are as follows: S1. The casein decapeptide was dissolved in PBS buffer to prepare a peptide solution at a concentration of 10 mg / mL. Polyethylene glycol was then added at a mass ratio of 1.5:1 between polyethylene glycol and casein decapeptide. The solution was stirred at room temperature for 12 h, dialyzed, and lyophilized to obtain a modified peptide powder. S2. The modified polypeptide powder was dissolved in a 3% CaCl2 solution to obtain a modified polypeptide solution having a concentration of 5 mg / mL; S3. Lactobacillus plantarum was cultured to the logarithmic phase, the cells were collected by centrifugation, washed with sterile PBS buffer, and resuspended to obtain a concentration of 1×10 9 CFU / mL of probiotic suspension; S4. The modified polypeptide solution and the probiotic suspension were mixed in a volume ratio of 2:1, the pH was adjusted to 7.0, the mixture was allowed to stand at 37°C for 4 hours, and freeze-dried to obtain the modified milk-derived active peptide carrying the probiotics. Example 3 A modified milk-derived active peptide carrying probiotics, the specific preparation steps are as follows: S1. Dissolve the casein decapeptide in PBS buffer to a concentration of 10 mg / mL. Add polyethylene glycol at a mass ratio of 2:1. Stir at room temperature for 12 h, dialyze, and lyophilize to obtain a modified polypeptide powder. S2. The modified polypeptide powder was dissolved in a 3% CaCl2 solution to obtain a modified polypeptide solution having a concentration of 5 mg / mL; S3. Lactobacillus plantarum was cultured to the logarithmic phase, the cells were collected by centrifugation, washed with sterile PBS buffer, and resuspended to obtain a concentration of 1×10 9 CFU / mL of probiotic suspension; S4. The modified polypeptide solution and the probiotic suspension were mixed in a volume ratio of 2:1, the pH was adjusted to 7.0, the mixture was allowed to stand at 37°C for 4 hours, and freeze-dried to obtain the modified milk-derived active peptide carrying the probiotics. Example 4 A modified milk-derived active peptide carrying probiotics, the specific preparation steps are as follows: S1. Dissolve the casein decapeptide in PBS buffer to a concentration of 10 mg / mL. Then add polyethylene glycol at a mass ratio of 2.5:1 to the casein decapeptide. Stir at room temperature for 12 h, dialyze, and lyophilize to obtain a modified polypeptide powder. S2. The modified polypeptide powder was dissolved in a 3% CaCl2 solution to obtain a modified polypeptide solution having a concentration of 5 mg / mL; S3. Lactobacillus plantarum was cultured to the logarithmic phase, the cells were collected by centrifugation, washed with sterile PBS buffer, and resuspended to obtain a concentration of 1×10 9 CFU / mL of probiotic suspension; S4. The modified polypeptide solution and the probiotic suspension were mixed in a volume ratio of 2:1, the pH was adjusted to 7.0, the mixture was allowed to stand at 37°C for 4 hours, and freeze-dried to obtain the modified milk-derived active peptide carrying the probiotics. Example 5 A modified milk-derived active peptide carrying probiotics, the specific preparation steps are as follows: S1. Dissolve the casein hydrolysate decapeptide in PBS buffer to a concentration of 10 mg / mL. Then add polyethylene glycol at a mass ratio of 3:1. Stir at room temperature for 12 h, dialyze, and lyophilize to obtain a modified polypeptide powder. S2. The modified polypeptide powder was dissolved in a 3% CaCl2 solution to obtain a modified polypeptide solution having a concentration of 5 mg / mL; S3. Lactobacillus plantarum was cultured to the logarithmic phase, the cells were collected by centrifugation, washed with sterile PBS buffer, and resuspended to obtain a concentration of 1×10 9 CFU / mL of probiotic suspension; S4. The modified polypeptide solution and the probiotic suspension were mixed in a volume ratio of 2:1, the pH was adjusted to 7.0, the mixture was allowed to stand at 37°C for 4 hours, and freeze-dried to obtain the modified milk-derived active peptide carrying the probiotics. Example 6 A modified milk-derived active peptide carrying probiotics, the specific preparation steps are as follows: S1. Dissolve the casein decapeptide in PBS buffer to a concentration of 10 mg / mL. Add polyethylene glycol at a mass ratio of 2:1. Stir at room temperature for 12 h, dialyze, and lyophilize to obtain a modified polypeptide powder. S2. The modified polypeptide powder was dissolved in a 3% CaCl2 solution to obtain a modified polypeptide solution having a concentration of 5 mg / mL; S3. Lactobacillus plantarum was cultured to the logarithmic phase, the cells were collected by centrifugation, washed with sterile PBS buffer, and resuspended to obtain a concentration of 1×10 8CFU / mL of probiotic suspension; S4. The modified polypeptide solution and the probiotic suspension were mixed in a volume ratio of 2:1, the pH was adjusted to 7.0, the mixture was allowed to stand at 37°C for 4 hours, and freeze-dried to obtain the modified milk-derived active peptide carrying the probiotics. Example 7 A modified milk-derived active peptide carrying probiotics, the specific preparation steps are as follows: S1. Dissolve the casein decapeptide in PBS buffer to a concentration of 10 mg / mL. Add polyethylene glycol at a mass ratio of 2:1. Stir at room temperature for 12 h, dialyze, and lyophilize to obtain a modified polypeptide powder. S2. The modified polypeptide powder was dissolved in a 3% CaCl2 solution to obtain a modified polypeptide solution having a concentration of 5 mg / mL; S3. Lactobacillus plantarum was cultured to the logarithmic phase, the cells were collected by centrifugation, washed with sterile PBS buffer, and resuspended to obtain a concentration of 1×10 9 CFU / mL of probiotic suspension; S4. The modified polypeptide solution and the probiotic suspension were mixed in a volume ratio of 1:1, the pH was adjusted to 7.0, the mixture was allowed to stand at 37°C for 4 hours, and freeze-dried to obtain the modified milk-derived active peptide carrying the probiotics. Example 8 A modified milk-derived active peptide carrying probiotics, the specific preparation steps are as follows: S1. Dissolve the casein decapeptide in PBS buffer to a concentration of 10 mg / mL. Add polyethylene glycol at a mass ratio of 2:1. Stir at room temperature for 12 h, dialyze, and lyophilize to obtain a modified polypeptide powder. S2. The modified polypeptide powder was dissolved in a 3% CaCl2 solution to obtain a modified polypeptide solution having a concentration of 5 mg / mL; S3. Lactobacillus plantarum was cultured to the logarithmic phase, the cells were collected by centrifugation, washed with sterile PBS buffer, and resuspended to obtain a concentration of 1×10 9 CFU / mL of probiotic suspension; S4. The modified polypeptide solution and the probiotic suspension were mixed at a volume ratio of 1.5:1, the pH was adjusted to 7.0, the mixture was allowed to stand at 37°C for 4 hours, and freeze-dried to obtain the modified milk-derived active peptide carrying the probiotics. Example 9 A modified milk-derived active peptide carrying probiotics, the specific preparation steps are as follows: S1. Dissolve the casein decapeptide in PBS buffer to a concentration of 10 mg / mL. Add polyethylene glycol at a mass ratio of 2:1. Stir at room temperature for 12 h, dialyze, and lyophilize to obtain a modified polypeptide powder. S2. The modified polypeptide powder was dissolved in a 3% CaCl2 solution to obtain a modified polypeptide solution having a concentration of 5 mg / mL; S3. Lactobacillus plantarum was cultured to the logarithmic phase, the cells were collected by centrifugation, washed with sterile PBS buffer, and resuspended to obtain a concentration of 1×10 9 CFU / mL of probiotic suspension; S4. The modified polypeptide solution and the probiotic suspension were mixed in a volume ratio of 2.5:1, the pH was adjusted to 7.0, the mixture was allowed to stand at 37°C for 4 hours, and freeze-dried to obtain the modified milk-derived active peptide carrying the probiotics. Comparative Example 1 The difference between this comparative example and Example 3 is that no polyethylene glycol modification was performed, as follows: A modified milk-derived active peptide carrying probiotics, the specific preparation steps are as follows: S1. Dissolve casein hydrolysate decapeptide powder in 3% CaCl2 solution to prepare a 5 mg / mL peptide solution; S2. Lactobacillus plantarum was cultured to the logarithmic phase, the cells were collected by centrifugation, washed with sterile PBS buffer, and resuspended to obtain a concentration of 1×10 9 CFU / mL of probiotic suspension; S3. Mix the polypeptide solution and the probiotic suspension in a volume ratio of 2:1, adjust the pH to 7.0, let it stand at 37°C for 4 hours, and freeze-dry to obtain the milk-derived active peptides carrying probiotics. Comparative Example 2 The difference between this comparative example and Example 3 is that the amount of polyethylene glycol added is too low, and the mass ratio of polyethylene glycol to casein hydrolysate decapeptide is 0.5:1, as follows: A modified milk-derived active peptide carrying probiotics, the specific preparation steps are as follows: S1. The casein decapeptide was dissolved in PBS buffer to prepare a peptide solution with a concentration of 10 mg / mL. Polyethylene glycol was then added at a mass ratio of 0.5:1 between polyethylene glycol and casein decapeptide. The solution was stirred at room temperature for 12 h, dialyzed, and lyophilized to obtain a modified peptide powder. S2. The modified polypeptide powder was dissolved in a 3% CaCl2 solution to obtain a modified polypeptide solution having a concentration of 5 mg / mL; S3. Lactobacillus plantarum was cultured to the logarithmic phase, the cells were collected by centrifugation, washed with sterile PBS buffer, and resuspended to obtain a concentration of 1×10 9CFU / mL of probiotic suspension; S4. The modified polypeptide solution and the probiotic suspension were mixed in a volume ratio of 2:1, the pH was adjusted to 7.0, the mixture was allowed to stand at 37°C for 4 hours, and freeze-dried to obtain the modified milk-derived active peptide carrying the probiotics. Comparative Example 3 The difference between this comparative example and Example 3 is that the amount of polyethylene glycol added is too high, and the mass ratio of polyethylene glycol to casein hydrolysate decapeptide is 4:1, as follows: A modified milk-derived active peptide carrying probiotics, the specific preparation steps are as follows: S1. Dissolve the casein decapeptide in PBS buffer to a concentration of 10 mg / mL. Then add polyethylene glycol at a mass ratio of 4:1. Stir at room temperature for 12 h, dialyze, and lyophilize to obtain a modified polypeptide powder. S2. The modified polypeptide powder was dissolved in a 3% CaCl2 solution to obtain a modified polypeptide solution having a concentration of 5 mg / mL; S3. Lactobacillus plantarum was cultured to the logarithmic phase, the cells were collected by centrifugation, washed with sterile PBS buffer, and resuspended to obtain a concentration of 1×10 9 CFU / mL of probiotic suspension; S4. The modified polypeptide solution and the probiotic suspension were mixed in a volume ratio of 2:1, the pH was adjusted to 7.0, the mixture was allowed to stand at 37°C for 4 hours, and freeze-dried to obtain the modified milk-derived active peptide carrying the probiotics. Comparative Example 4 The difference between this comparative example and Example 3 is that the probiotics are not embedded, as follows: A modified milk-derived active peptide carrying probiotics, the specific preparation steps are as follows: S1. Dissolve the casein decapeptide in PBS buffer to a concentration of 10 mg / mL. Add polyethylene glycol at a mass ratio of 2:1. Stir at room temperature for 12 h, dialyze, and lyophilize to obtain a modified polypeptide powder. S2. The modified polypeptide powder was dissolved in a 3% CaCl2 solution to obtain a 5 mg / mL modified polypeptide solution. The solution was adjusted to pH 7.0, allowed to stand at 37°C for 4 h, and freeze-dried to obtain the modified milk-derived active peptide. Comparative Example 5 The difference between this comparative example and Example 3 is that the volume ratio of the modified polypeptide solution to the probiotic suspension is too low, which is 0.5:1, as follows: A modified milk-derived active peptide carrying probiotics, the specific preparation steps are as follows: S1. Dissolve the casein decapeptide in PBS buffer to a concentration of 10 mg / mL. Add polyethylene glycol at a mass ratio of 2:1. Stir at room temperature for 12 h, dialyze, and lyophilize to obtain a modified polypeptide powder. S2. The modified polypeptide powder was dissolved in a 3% CaCl2 solution to obtain a modified polypeptide solution having a concentration of 5 mg / mL; S3. Lactobacillus plantarum was cultured to the logarithmic phase, the cells were collected by centrifugation, washed with sterile PBS buffer, and resuspended to obtain a concentration of 1×10 9 CFU / mL of probiotic suspension; S4. The modified polypeptide solution and the probiotic suspension were mixed at a volume ratio of 0.5:1, the pH was adjusted to 7.0, the mixture was allowed to stand at 37°C for 4 hours, and freeze-dried to obtain the modified milk-derived active peptide carrying the probiotics. Comparative Example 6 The difference between this comparative example and Example 3 is that the volume ratio of the modified polypeptide solution to the probiotic suspension is too high, which is 3:1. The specific steps are as follows: A modified milk-derived active peptide carrying probiotics is prepared as follows: S1. Dissolve the casein decapeptide in PBS buffer to a concentration of 10 mg / mL. Add polyethylene glycol at a mass ratio of 2:1. Stir at room temperature for 12 h, dialyze, and lyophilize to obtain a modified polypeptide powder. S2. The modified polypeptide powder was dissolved in a 3% CaCl2 solution to obtain a modified polypeptide solution having a concentration of 5 mg / mL; S3. Lactobacillus plantarum was cultured to the logarithmic phase, the cells were collected by centrifugation, washed with sterile PBS buffer, and resuspended to obtain a concentration of 1×10 9 CFU / mL of probiotic suspension; S4. The modified polypeptide solution and the probiotic suspension were mixed in a volume ratio of 3:1, the pH was adjusted to 7.0, the mixture was allowed to stand at 37°C for 4 hours, and freeze-dried to obtain the modified milk-derived active peptide carrying the probiotics. Comparative Example 7 The difference between this comparative example and Example 3 is that no CaCl2 was added, as follows: A modified milk-derived active peptide carrying probiotics, the specific preparation steps are as follows: S1. Dissolve the casein decapeptide in PBS buffer to a concentration of 10 mg / mL. Add polyethylene glycol at a mass ratio of 2:1. Stir at room temperature for 12 h, dialyze, and lyophilize to obtain a modified polypeptide powder. S2. The modified polypeptide powder was dissolved in water to obtain a modified polypeptide solution having a concentration of 5 mg / mL; S3. Lactobacillus plantarum was cultured to the logarithmic phase, the cells were collected by centrifugation, washed with sterile PBS buffer, and resuspended to obtain a concentration of 1×10 9 CFU / mL of probiotic suspension; S4. The modified polypeptide solution and the probiotic suspension were mixed in a volume ratio of 2:1, the pH was adjusted to 7.0, the mixture was allowed to stand at 37°C for 4 hours, and freeze-dried to obtain the modified milk-derived active peptide carrying the probiotics. Performance testing: (1) Probiotic encapsulation rate Table 1 Probiotic embedding rate in modified milk-derived active peptides prepared in Examples 1-9 and Comparative Examples 1-7 As shown in Table 1, the probiotic embedding rate in the modified milk-derived active peptides prepared in Examples 1-9 is 80.5-85.6%, among which the probiotic embedding rate in Example 3 is the highest at 85.6%. 2+ The stability and structure of the prepared modified milk-derived active peptides are relatively poor, so the encapsulation rate of probiotics will also decrease accordingly. (2) Probiotic release rate The modified milk-derived active peptides prepared in the examples and comparative examples were placed in simulated gastric fluid, incubated at 37°C with shaking for 4 hours, and sampled every 2 hours; the sample treated with gastric fluid was transferred to simulated intestinal fluid, incubated at 37°C with continuous shaking for 4 hours, and sampled every 2 hours. The released liquid was gradiently diluted and applied to the culture medium, and the number of released viable bacteria was calculated. The probiotic release rate was calculated according to the following formula: Probiotic release rate (%) = number of live bacteria released in the stomach or intestine / number of live bacteria initially embedded × 100% Table 2 Probiotic release rate of modified milk-derived active peptides prepared in Examples 1-9 and Comparative Examples 1-7 As shown in Table 2, the modified milk-derived active peptide prepared in Example 3 has a low release rate of 14.2% in the stomach, while the release rate of probiotics in the intestine is the highest at 83.6%. 2+ The polypeptide hydrogel structure produced by self-assembly is more stable, which can well protect the probiotics in the stomach and enable them to be efficiently released in the intestine. In summary, the modified milk-derived active peptide prepared in Example 3 was selected and applied to the subsequent preparation of the sleep-aiding milk beverage. Example 10 The application of modified milk-derived active peptides carrying probiotics in sleep-aiding milk beverages is as follows: (1) Mixing: 1% cream, 0.1% γ-aminobutyric acid, 0.1% jujube kernel powder, 0.05% yam powder, 0.002% edible salt, 0.2% emulsifier stabilizer, 1.8% erythritol, 0.001% sucralose, 0.01% sodium carbonate, and 4% of the modified milk-derived active peptide carrying probiotics prepared in Example 3 were mixed, and sheared in 80°C hot water for 15 minutes to fully dissolve it. Then, 68% raw milk was added and pre-filled with softened water to 85% of the required liquid level. The mixture was stirred for 10 minutes to mix the material and the liquid evenly. Then, 0.04% edible flavor was added and the volume was fixed. After the volume was fixed, the mixture was stirred for 5 minutes to mix the material and the liquid evenly. (2) Homogenization and sterilization: First, homogenize at a pressure of 20 MPa and a temperature of 60°C for 15 seconds; after the liquid is cooled to 15°C, perform ultra-high temperature instantaneous sterilization at a pressure of 28 MPa and a temperature of 65°C for 4 seconds; after sterilization, cool to 30°C and wait for filling; (3) Aseptic canning: Under the filling temperature of 30℃, the liquid is aseptically filled to obtain the sleep-aiding milk beverage. Comparative Example 8 The difference between this comparative example and Example 10 is that no modified milk-derived active peptide is added, specifically as follows: (1) Mixing: 1% cream, 0.1% γ-aminobutyric acid, 0.1% jujube kernel powder, 0.05% yam powder, 0.002% edible salt, 0.2% emulsifier stabilizer, 1.8% erythritol, 0.001% sucralose, and 0.01% sodium carbonate were mixed and sheared in 80°C hot water for 15 minutes to fully dissolve them. Then, 68% raw milk was added and the volume was pre-filled to 85% of the required liquid level with softened water. The mixture was stirred for 10 minutes to mix the mixture evenly. Then, 0.04% edible flavor was added and the volume was fixed. After the volume was fixed, the mixture was stirred for 5 minutes to mix the mixture evenly. (2) Homogenization and sterilization: First, homogenize at a pressure of 20 MPa and a temperature of 60°C for 15 seconds; after the liquid is cooled to 15°C, perform ultra-high temperature instantaneous sterilization at a pressure of 28 MPa and a temperature of 65°C for 4 seconds; after sterilization, cool to 30°C and wait for filling; (3) Aseptic canning: Under the filling temperature of 30℃, the liquid is aseptically filled to obtain the sleep-aiding milk beverage. Comparative Example 9 The difference between this comparative example and Example 10 is that milk-derived active peptides are directly added, as follows: (1) Mixing: 1% cream, 0.1% γ-aminobutyric acid, 0.1% jujube kernel powder, 0.05% yam powder, 0.002% edible salt, 0.2% emulsifier stabilizer, 1.8% erythritol, 0.001% sucralose, 0.01% sodium carbonate, and 4% casein hydrolysate decapeptide were mixed, and sheared in 80°C hot water for 15 minutes to fully dissolve them. Then, 68% raw milk was added and the volume was pre-filled to 85% of the required liquid level with softened water. The mixture was stirred for 10 minutes to mix the mixture evenly. Then, 0.04% edible flavor was added and the volume was fixed. After the volume was fixed, the mixture was stirred for 5 minutes to mix the mixture evenly. (2) Homogenization and sterilization: First, homogenize at a pressure of 20 MPa and a temperature of 60°C for 15 seconds; after the liquid is cooled to 15°C, perform ultra-high temperature instantaneous sterilization at a pressure of 28 MPa and a temperature of 65°C for 4 seconds; after sterilization, cool to 30°C and wait for filling; (3) Aseptic canning: Under the filling temperature of 30℃, the liquid is aseptically filled to obtain the sleep-aiding milk beverage. Comparative Example 10 The difference between this comparative example and Example 10 is that milk-derived active peptides and probiotics are directly added at the same time, as follows: (1) Mixing: 1% cream, 0.1% γ-aminobutyric acid, 0.1% jujube kernel powder, 0.05% yam powder, 0.002% edible salt, 0.2% emulsifier stabilizer, 1.8% erythritol, 0.001% sucralose, 0.01% sodium carbonate, 3% casein hydrolysate decapeptide, and 1% Lactobacillus plantarum are mixed and sheared in 80°C hot water for 15 minutes to fully dissolve the mixture. Then, 68% raw milk is added and the mixture is pre-filled to 85% of the required liquid level with softened water. The mixture is stirred for 10 minutes to mix the mixture evenly. Then, 0.04% edible flavor is added and the mixture is fixed to volume. After the volume is fixed, the mixture is stirred for 5 minutes to mix the mixture evenly. (2) Homogenization and sterilization: First, homogenize at a pressure of 20 MPa and a temperature of 60°C for 15 seconds; after the liquid is cooled to 15°C, perform ultra-high temperature instantaneous sterilization at a pressure of 28 MPa and a temperature of 65°C for 4 seconds; after sterilization, cool to 30°C and wait for filling; (3) Aseptic canning: Under the filling temperature of 30℃, the liquid is aseptically filled to obtain the sleep-aiding milk beverage. Comparative Example 11 The difference between this comparative example and Example 10 is that the modified milk-derived active peptide prepared in Comparative Example 1 was used. Comparative Example 12 The difference between this comparative example and Example 10 is that the modified milk-derived active peptide prepared in Comparative Example 2 was used. Comparative Example 13 The difference between this comparative example and Example 10 is that the modified milk-derived active peptide prepared in Comparative Example 3 was used. Comparative Example 14 The difference between this comparative example and Example 10 is that the modified milk-derived active peptide prepared in Comparative Example 4 was used. Comparative Example 15 The difference between this comparative example and Example 10 is that the modified milk-derived active peptide prepared in Comparative Example 5 is used. Comparative Example 16 The difference between this comparative example and Example 10 is that the modified milk-derived active peptide prepared in Comparative Example 6 is used. Comparative Example 17 The difference between this comparative example and Example 10 is that the modified milk-derived active peptide prepared in Comparative Example 7 is used. (1) Drinking effect evaluation The sleep-aiding milk beverages prepared in Example 10 and Comparative Examples 8-17 were tasted and investigated by 100 evaluators. The beverages were consumed for 30 consecutive days to determine their sleep-improving effects. The results are shown in Table 3. Table 3 Evaluation table of drinking effects of sleep-aiding milk beverages prepared in Example 10 and Comparative Examples 8-17 The data in Table 3 show that the sleep-aiding milk beverage prepared in Example 10 is superior to the other comparative examples in terms of overall flavor and sleep-promoting effect. It can be seen that the addition of the modified milk-derived active peptides encapsulating probiotics is more helpful in improving the sleep effect. (2) Evaluation of sleep-aiding effects 1. Animal Grouping and Establishment of Insomnia Mouse Model All mice (weighing 20 ± 3 g) were specific pathogen-free. Healthy female SPF Kunming mice were provided by the Shanghai Laboratory Animal Center and maintained on a regular pelleted rodent diet and purified water. All procedures involving mice were in accordance with the guidelines provided by the Shanghai Laboratory Animal Care and Animal Experimentation Center. A total of 96 mice (8 mice / group) were randomly selected, and 88 mice were used to establish an insomnia model using a modified horizontal turntable sleep deprivation method. A sleep deprivation box with a length of 110 cm, a width of 60 cm, and a height of 40 cm was constructed using acrylic plastic plates. With a standard longitudinal distance of 10 cm and a horizontal distance of 13 cm, 15 cylindrical platforms with a height of 8 cm and a diameter of 6.5 cm were fixed at the bottom. Water at 23-25°C was injected to 1 cm below the platform to maintain the water temperature. After 7 days of adaptive training, the modeling mice were trained to stand and placed on the cylindrical platform. When the mice were about to fall asleep, they were awakened by their muscles relaxing and their heads touching the water or falling into the water, forcing them to stay awake and stand. During the 2-week modeling period, the mice rested once in the morning and evening for 30 minutes each time. Behavioral changes in the mice, including changes in their mental state and diet, were recorded every 2 days. 88 insomnia model mice were randomly divided into 11 groups, namely Example 10 group, Comparative Example 8 group, Comparative Example 9 group, Comparative Example 10 group, Comparative Example 11 group, Comparative Example 12 group, Comparative Example 13 group, Comparative Example 14 group, Comparative Example 15 group, Comparative Example 16 group, Comparative Example 17 group. The remaining 8 mice without modeling were used as a control group. At the beginning of the second week of standing training, they were gavage-treated with a sleep-aiding milk drink, and the control group mice were gavage-treated with an equal volume of purified water. 12 hours after the last gavage (4 weeks of intervention with a sleep-aiding milk drink), the mice were anesthetized and killed by an overdose of sodium pentobarbital. 2. Sleep improvement test All tests were conducted in a quiet environment and were carried out one day before the end of the experiment. The details are as follows: a. Pentobarbital-induced sleep duration test: During the sleep test, sleep and wakefulness were determined based on the loss of the righting reflex. Loss of the righting reflex for more than 30 seconds was considered sleep. Thirty minutes after gavage, each group of mice received intraperitoneal administration of sodium pentobarbital (49 mg / kg / bw). Sleep duration was recorded as the interval from loss of the righting reflex to recovery. Depend on Figure 1 It can be seen that compared with the control group, the symptoms of the insomnia mice in group 10 of Example 10 of the present invention that were gavage-administered were alleviated, and the sleep time was significantly prolonged, while the sleep time was the shortest in group 8 of the comparative example in which no modified milk-derived active peptide was added; and the effect of improving sleep in group 14 of the comparative example in which probiotics were lacking was not significant. b. Pentobarbital-induced sleep latency test: Each mouse was intraperitoneally injected with 320 mg / kg / bw of pentobarbital sodium for 30 minutes. Sleep latency was recorded as the interval from pentobarbital injection to loss of righting reflex. Depend on Figure 2 It can be seen that compared with the control group, the sleep latency of the insomnia mice gavaged in Example 10 of the present invention was significantly shortened, and the insomnia mice were easier to fall asleep; while the sleep latency of the comparative example 8 group was prolonged in the absence of modified milk-derived active peptides; the sleep latency of the comparative example 14 group was also longer in the absence of probiotics, and the sleep disorder of the insomnia mice was still more serious. 3. Determination of brain 5-HT content: After the mice were sacrificed, the whole brain was removed by craniotomy in an ice bath. The brain was separated on an ice plate, the blood was rinsed with 0.9% ice sodium chloride solution, and the brain was dried with filter paper. After the brain was weighed, 9 times the amount of 0.9% ice NaCl solution was added and homogenized at 4°C. The slurry was transferred to a 2 mL centrifuge tube and centrifuged at 3000 rpm for 15 min at 4°C. The supernatant was collected and the 5-HT content was determined according to the relevant operating procedures in the kit (purchased from Wuhan Pure Biotechnology Co., Ltd.). Depend on Figure 3It can be seen that compared with the control group, the 5-hydroxytryptamine content of the insomnia mice in the 10 groups of Examples of the present invention was significantly increased, and was also higher than that of the comparative example group. The modified milk-derived active peptide prepared by the present invention also encapsulates probiotics. The porous structure of the polypeptide hydrogel can have a sustained-release effect on the probiotics, so that the delivered probiotics can regulate the production of 5-hydroxytryptamine in the intestinal environment, and can enter the nerve cells with the blood, bind to the 5-hydroxytryptamine receptors between neurons, and play a role in regulating emotions, thereby preventing sleep disorders, improving sleep quality, and alleviating anxiety. The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Any person skilled in the art can, without departing from the spirit and technical solution of the present invention, use the methods and technical contents disclosed above to make many possible changes and modifications to the technical solution of the present invention, or modify it into an equivalent embodiment with equivalent changes. Therefore, any simple modification, equivalent replacement, equivalent change and modification made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention shall still fall within the scope of protection of the technical solution of the present invention.

Claims

1. A modified milk-derived active peptide carrying probiotics, characterized by: The modified milk-derived active peptide is obtained by self-assembling the modified milk-derived active peptide to form a hydrogel and embedding the probiotics; The modified milk-derived active peptide is obtained by introducing polyethylene glycol into the casein hydrolysate decapeptide YLGYLEQLLR.

2. The method for preparing a modified milk-derived active peptide carrying probiotics according to claim 1, characterized in that: The specific preparation steps are as follows: S1. Dissolve the casein hydrolysate decapeptide in PBS buffer, add polyethylene glycol, stir at room temperature for 10-12 hours, dialyze, and lyophilize to obtain a modified polypeptide powder; S2. The modified polypeptide powder was dissolved in a CaCl2 solution at a concentration of 2-5% to obtain a modified polypeptide solution; S3. The probiotics were cultured to the logarithmic phase, the cells were collected by centrifugation, washed with sterile PBS buffer, and resuspended to prepare a probiotic suspension; S4. Mix the modified polypeptide solution and the probiotic suspension, adjust the pH to 6.5-7.5, let stand at 30-37°C for 2-5 hours, and freeze-dry to obtain the modified milk-derived active peptide carrying probiotics.

3. The method for preparing a modified milk-derived active peptide carrying probiotics according to claim 2, characterized in that: The concentration of the casein hydrolysate decapeptide in step S1 is 10-15 mg / mL; the mass ratio of the polyethylene glycol to the casein hydrolysate decapeptide is (1-3):

1.

4. The method for preparing a modified milk-derived active peptide carrying probiotics according to claim 2, characterized in that: The concentration of the modified polypeptide solution in step S2 is 2-10 mg / mL.

5. The method for preparing a modified milk-derived active peptide carrying probiotics according to claim 2, characterized in that: The probiotics in step S3 are any one of Lactobacillus plantarum, Bifidobacterium, and Lactobacillus fermentum; the concentration of the probiotic suspension is 1×10 8 -1×10 9 CFU / mL.

6. The method for preparing a modified milk-derived active peptide carrying probiotics according to claim 2, characterized in that: In step S4, the volume ratio of the modified polypeptide solution to the probiotic suspension is (1-2.5):

1.

7. Use of the modified milk-derived active peptide carrying probiotics prepared by the preparation method according to any one of claims 2 to 6 in a sleep-aiding milk beverage.

Images