Modified milk-derived active peptide carrying probiotics and application thereof in sleep-aiding milk beverage

Abstract

The application provides a modified milk source active peptide carrying probiotics and application of the modified milk source active peptide in sleep-aiding milk beverages, a modified polypeptide is prepared by introducing polyethylene glycol into casein proteolysis product decapetide YLGYLEQLLR, the modified polypeptide is self-assembled to form a hydrogel, and probiotics are embedded to obtain the modified milk source active peptide carrying probiotics. In the application, the modified milk source active peptide can embed and release the probiotics, so that the probiotics are continuously released in the body, and the sleep quality is improved; in addition, the probiotics and the polypeptide synergistically act to regulate the mood and improve the sleep quality.

Description

Technical Field

[0001] This invention belongs to the field of dairy processing technology, specifically relating to a modified milk-derived bioactive peptide carrying probiotics and its application in sleep-aiding milk beverages. Background Technology

[0002] With the fast pace of life and increasing work pressure, more and more people are facing sleep problems. According to relevant statistics, about one-third of the world's population suffers from sleep problems, with sleep deprivation being the most prominent. Sleep problems not only affect people's daily lives and work efficiency, but are also accompanied by other issues such as anxiety and depression.

[0003] Most sleep aids currently on the market are pharmaceutical products, primarily antidepressants and melatonin-based drugs. Long-term use can lead to drug dependence and may cause serious sleep disorders. Meanwhile, some health supplements have limited effectiveness and do not provide any benefit to the patient's body.

[0004] Existing research indicates that probiotics can regulate host neurotransmitter levels (such as serotonin) 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 significantly reduced number of live bacteria reaching the intestines (survival rate <30%), severely limiting their practical application. Furthermore, free probiotics have a short colonization time in the gut, making it impossible to continuously release active ingredients for long-term regulatory effects, necessitating the development of novel protected delivery systems.

[0005] YLGYLEQLLR, a decapeptide with sleep-aiding properties, has been identified among casein protein hydrolysates. 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 gastric environment, leading to loss of activity. Therefore, it is necessary to develop a system that modifies peptides and encapsulates probiotics for application in the field of sleep-aid foods. Summary of the Invention

[0006] Technical Problem to be Solved: To address the aforementioned technical problems, the purpose of this invention is to provide a modified milk-derived bioactive peptide incorporating probiotics and its application in sleep-aiding milk beverages. A modified polypeptide is prepared by introducing polyethylene glycol onto the casein hydrolysate decapeptide YLGYLEQLLR. Based on the self-assembly of the modified milk-derived bioactive peptide, a hydrogel is formed, and probiotics are encapsulated to obtain a modified milk-derived bioactive peptide incorporating probiotics. This modified milk-derived bioactive peptide encapsulates and slowly releases probiotics, allowing for continuous release of probiotics in the body, thus improving sleep quality. Furthermore, the probiotics and polypeptides work synergistically to regulate mood and improve sleep quality.

[0007] Technical solution: A modified milk-derived active peptide carrying probiotics, wherein the modified milk-derived active peptide is obtained by self-assembling the modified milk-derived active peptide into a hydrogel and encapsulating probiotics;

[0008] The modified milk-derived bioactive peptide is obtained by introducing polyethylene glycol onto the casein hydrolysate decapeptide YLGYLEQLLR. The specific preparation steps of the above-mentioned method for preparing a probiotic-loaded modified milk-derived bioactive peptide are as follows:

[0009] S1. Dissolve the casein hydrolysate decapeptide in PBS buffer, add polyethylene glycol, stir at room temperature for 10-12 h, dialyze, and freeze dry to obtain modified peptide powder.

[0010] S2. Dissolve the modified polypeptide powder in a 2-5% CaCl2 solution to obtain a modified polypeptide solution;

[0011] S3. Cultivate probiotics to the logarithmic phase, collect the bacterial cells by centrifugation, wash with sterile PBS buffer, resuspend, and obtain a probiotic suspension; S4. Mix the modified peptide solution and the probiotic suspension, adjust the pH to 6.5-7.5, let stand at 30-37℃ for 2-5 hours, freeze dry, and obtain the modified milk-derived active peptides loaded with probiotics.

[0012] Furthermore, in step S1, the concentration of casein hydrolysate decapeptide is 10-15 mg / mL; the mass ratio of polyethylene glycol to casein hydrolysate decapeptide is (1-3):1.

[0013] Furthermore, the concentration of the modified polypeptide solution in step S2 is 2-10 mg / mL.

[0014] Furthermore, in step S3, the probiotics are any one of Lactobacillus plantarum, Bifidobacterium, and Lactobacillus fermentum; the concentration of the probiotic suspension is 1×10⁻⁶. 8 -1×10 9 CFU / mL.

[0015] Furthermore, in step S4, the volume ratio of the modified polypeptide solution to the probiotic suspension is (1-2.5):1.

[0016] The application of the modified milk-derived bioactive peptides containing probiotics prepared by the above method in sleep-aiding milk beverages.

[0017] Furthermore, the formula of the sleep-aiding milk beverage is as follows (by weight percentage): 65-70% raw milk; 1-2% light cream; 0.05-0.2% γ-aminobutyric acid; 0.05-0.2% jujube seed powder; 0.02-1% yam powder; 0-0.005% edible salt; 0.1-0.5% emulsifying stabilizer; 1-5% erythritol; 0-0.005% sucralose; 0-0.03% sodium carbonate; 0-0.07% edible flavoring; and 2-5% modified milk-derived active peptides infused with probiotics.

[0018] Beneficial effects:

[0019] 1. The modified milk-derived active peptide prepared in this invention is based on decapeptide from casein hydrolysate. Decapeptide YLGYLEQLLR has a sleep-aiding function and can bind to neuronal 5-hydroxytryptamine receptors across the blood-brain barrier to directly regulate 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 thus failing to effectively encapsulate probiotics and deliver them to the intestines. Therefore, this invention modifies the decapeptide from casein hydrolysate by polyethylene glycol grafting. Polyethylene glycol covalently binds to the amino or carboxyl groups of the peptide, forming steric hindrance, which can reduce the contact between gastric acid and digestive enzymes and the peptide, and increase the stability of the peptide hydrogel in the stomach.

[0020] 2. To further improve the stability of the peptide in the stomach, this invention also adds Ca during the preparation of the modified peptide self-assembled hydrogel. 2+ Calcium ions can bind to the carboxyl groups on casein peptide chains through electrostatic interactions, forming salt bridges, thereby enhancing the cross-linking between peptide chains and improving the stability of polypeptide hydrogels.

[0021] 3. The modified milk-derived active peptides prepared in this invention also contain probiotics. The porous structure of the polypeptide hydrogel provides sustained release for the probiotics, enabling the delivered probiotics to regulate the production of serotonin in the intestinal environment and enter nerve cells via the bloodstream. They then bind to serotonin receptors between neurons, thereby regulating mood and preventing sleep disorders, improving sleep quality, and relieving anxiety. Attached Figure Description

[0022] Figure 1 Sleep duration graphs for insomnia mice in the control group, Example 10 group, and Comparative Examples 8-15;

[0023] Figure 2 Sleep latency diagrams for insomnia mice in the control group, Example 10 group, and Comparative Examples 8-15 group;

[0024] Figure 3 The graph shows the 5-hydroxytryptamine content in the brain tissue of insomnia mice in the control group, Example 10 group, and Comparative Examples 8-15. Detailed Implementation

[0025] The present invention will be further described below with reference to the accompanying drawings and embodiments. The following embodiments are illustrative of the present invention, but the present invention is not limited to the following embodiments:

[0026] Example 1

[0027] The specific preparation steps of a modified milk-derived bioactive peptide carrying probiotics are as follows:

[0028] S1. Dissolve the casein hydrolysate decapeptide in PBS buffer to prepare a polypeptide solution with a concentration of 10 mg / mL. Then add polyethylene glycol, with a mass ratio of polyethylene glycol to casein hydrolysate decapeptide of 1:1. Stir at room temperature for 12 h, dialyze, and freeze dry to obtain modified polypeptide powder.

[0029] S2. Dissolve the modified polypeptide powder in a 3% CaCl2 solution to obtain a modified polypeptide solution with a concentration of 5 mg / mL;

[0030] S3. Culture *Lactobacillus plantarum* to the logarithmic growth phase, collect the cells by centrifugation, wash with sterile PBS buffer, resuspend, and prepare a concentration of 1×10⁻⁶. 9 CFU / mL probiotic suspension;

[0031] S4. Mix the modified peptide solution and probiotic suspension at a volume ratio of 2:1, adjust the pH to 7.0, let stand at 37°C for 4 hours, and freeze dry to obtain the modified milk-derived active peptides loaded with probiotics.

[0032] Example 2

[0033] The specific preparation steps of a modified milk-derived bioactive peptide carrying probiotics are as follows:

[0034] S1. Dissolve the casein hydrolysate decapeptide in PBS buffer to prepare a polypeptide solution with a concentration of 10 mg / mL. Then add polyethylene glycol, with a mass ratio of polyethylene glycol to casein hydrolysate decapeptide of 1.5:1. Stir at room temperature for 12 h, dialyze, and freeze dry to obtain modified polypeptide powder.

[0035] S2. Dissolve the modified polypeptide powder in a 3% CaCl2 solution to obtain a modified polypeptide solution with a concentration of 5 mg / mL;

[0036] S3. Culture *Lactobacillus plantarum* to the logarithmic growth phase, collect the cells by centrifugation, wash with sterile PBS buffer, resuspend, and prepare a concentration of 1×10⁻⁶. 9 CFU / mL probiotic suspension;

[0037] S4. Mix the modified peptide solution and probiotic suspension at a volume ratio of 2:1, adjust the pH to 7.0, let stand at 37°C for 4 hours, and freeze dry to obtain the modified milk-derived active peptides loaded with probiotics.

[0038] Example 3

[0039] The specific preparation steps of a modified milk-derived bioactive peptide carrying probiotics are as follows:

[0040] S1. Dissolve the casein hydrolysate decapeptide in PBS buffer to prepare a polypeptide solution with a concentration of 10 mg / mL. Then add polyethylene glycol, with a mass ratio of polyethylene glycol to casein hydrolysate decapeptide of 2:1. Stir at room temperature for 12 h, dialyze, and freeze dry to obtain modified polypeptide powder.

[0041] S2. Dissolve the modified polypeptide powder in a 3% CaCl2 solution to obtain a modified polypeptide solution with a concentration of 5 mg / mL;

[0042] S3. Culture *Lactobacillus plantarum* to the logarithmic growth phase, collect the cells by centrifugation, wash with sterile PBS buffer, resuspend, and prepare a concentration of 1×10⁻⁶. 9 CFU / mL probiotic suspension;

[0043] S4. Mix the modified peptide solution and probiotic suspension at a volume ratio of 2:1, adjust the pH to 7.0, let stand at 37°C for 4 hours, and freeze dry to obtain the modified milk-derived active peptides loaded with probiotics.

[0044] Example 4

[0045] The specific preparation steps of a modified milk-derived bioactive peptide carrying probiotics are as follows:

[0046] S1. Dissolve the casein hydrolysate decapeptide in PBS buffer to prepare a polypeptide solution with a concentration of 10 mg / mL. Then add polyethylene glycol, with a mass ratio of polyethylene glycol to casein hydrolysate decapeptide of 2.5:1. Stir at room temperature for 12 h, dialyze, and freeze dry to obtain modified polypeptide powder.

[0047] S2. Dissolve the modified polypeptide powder in a 3% CaCl2 solution to obtain a modified polypeptide solution with a concentration of 5 mg / mL;

[0048] S3. Culture *Lactobacillus plantarum* to the logarithmic growth phase, collect the cells by centrifugation, wash with sterile PBS buffer, resuspend, and prepare a concentration of 1×10⁻⁶. 9 CFU / mL probiotic suspension;

[0049] S4. Mix the modified peptide solution and probiotic suspension at a volume ratio of 2:1, adjust the pH to 7.0, let stand at 37°C for 4 hours, and freeze dry to obtain the modified milk-derived active peptides loaded with probiotics.

[0050] Example 5

[0051] The specific preparation steps of a modified milk-derived bioactive peptide carrying probiotics are as follows:

[0052] S1. Dissolve the casein hydrolysate decapeptide in PBS buffer to prepare a polypeptide solution with a concentration of 10 mg / mL. Then add polyethylene glycol, with a mass ratio of polyethylene glycol to casein hydrolysate decapeptide of 3:1. Stir at room temperature for 12 h, dialyze, and freeze dry to obtain modified polypeptide powder.

[0053] S2. Dissolve the modified polypeptide powder in a 3% CaCl2 solution to obtain a modified polypeptide solution with a concentration of 5 mg / mL;

[0054] S3. Culture *Lactobacillus plantarum* to the logarithmic growth phase, collect the cells by centrifugation, wash with sterile PBS buffer, resuspend, and prepare a concentration of 1×10⁻⁶. 9 CFU / mL probiotic suspension;

[0055] S4. Mix the modified peptide solution and probiotic suspension at a volume ratio of 2:1, adjust the pH to 7.0, let stand at 37°C for 4 hours, and freeze dry to obtain the modified milk-derived active peptides loaded with probiotics.

[0056] Example 6

[0057] The specific preparation steps of a modified milk-derived bioactive peptide carrying probiotics are as follows:

[0058] S1. Dissolve the casein hydrolysate decapeptide in PBS buffer to prepare a polypeptide solution with a concentration of 10 mg / mL. Then add polyethylene glycol, with a mass ratio of polyethylene glycol to casein hydrolysate decapeptide of 2:1. Stir at room temperature for 12 h, dialyze, and freeze dry to obtain modified polypeptide powder.

[0059] S2. Dissolve the modified polypeptide powder in a 3% CaCl2 solution to obtain a modified polypeptide solution with a concentration of 5 mg / mL;

[0060] S3. Culture *Lactobacillus plantarum* to the logarithmic growth phase, collect the cells by centrifugation, wash with sterile PBS buffer, resuspend, and prepare a concentration of 1×10⁻⁶. 8 CFU / mL probiotic suspension;

[0061] S4. Mix the modified peptide solution and probiotic suspension at a volume ratio of 2:1, adjust the pH to 7.0, let stand at 37°C for 4 hours, and freeze dry to obtain the modified milk-derived active peptides loaded with probiotics.

[0062] Example 7

[0063] The specific preparation steps of a modified milk-derived bioactive peptide carrying probiotics are as follows:

[0064] S1. Dissolve the casein hydrolysate decapeptide in PBS buffer to prepare a polypeptide solution with a concentration of 10 mg / mL. Then add polyethylene glycol, with a mass ratio of polyethylene glycol to casein hydrolysate decapeptide of 2:1. Stir at room temperature for 12 h, dialyze, and freeze dry to obtain modified polypeptide powder.

[0065] S2. Dissolve the modified polypeptide powder in a 3% CaCl2 solution to obtain a modified polypeptide solution with a concentration of 5 mg / mL;

[0066] S3. Culture *Lactobacillus plantarum* to the logarithmic growth phase, collect the cells by centrifugation, wash with sterile PBS buffer, resuspend, and prepare a concentration of 1×10⁻⁶. 9 CFU / mL probiotic suspension;

[0067] S4. Mix the modified peptide solution and probiotic suspension at a volume ratio of 1:1, adjust the pH to 7.0, let stand at 37°C for 4 hours, and freeze dry to obtain the modified milk-derived active peptides loaded with probiotics.

[0068] Example 8

[0069] The specific preparation steps of a modified milk-derived bioactive peptide carrying probiotics are as follows:

[0070] S1. Dissolve the casein hydrolysate decapeptide in PBS buffer to prepare a polypeptide solution with a concentration of 10 mg / mL. Then add polyethylene glycol, with a mass ratio of polyethylene glycol to casein hydrolysate decapeptide of 2:1. Stir at room temperature for 12 h, dialyze, and freeze dry to obtain modified polypeptide powder.

[0071] S2. Dissolve the modified polypeptide powder in a 3% CaCl2 solution to obtain a modified polypeptide solution with a concentration of 5 mg / mL;

[0072] S3. Culture *Lactobacillus plantarum* to the logarithmic growth phase, collect the cells by centrifugation, wash with sterile PBS buffer, resuspend, and prepare a concentration of 1×10⁻⁶. 9 CFU / mL probiotic suspension;

[0073] S4. Mix the modified peptide solution and probiotic suspension at a volume ratio of 1.5:1, adjust the pH to 7.0, let stand at 37°C for 4 hours, and freeze dry to obtain the modified milk-derived active peptide loaded with probiotics.

[0074] Example 9

[0075] The specific preparation steps of a modified milk-derived bioactive peptide carrying probiotics are as follows:

[0076] S1. Dissolve the casein hydrolysate decapeptide in PBS buffer to prepare a polypeptide solution with a concentration of 10 mg / mL. Then add polyethylene glycol, with a mass ratio of polyethylene glycol to casein hydrolysate decapeptide of 2:1. Stir at room temperature for 12 h, dialyze, and freeze dry to obtain modified polypeptide powder.

[0077] S2. Dissolve the modified polypeptide powder in a 3% CaCl2 solution to obtain a modified polypeptide solution with a concentration of 5 mg / mL;

[0078] S3. Culture *Lactobacillus plantarum* to the logarithmic growth phase, collect the cells by centrifugation, wash with sterile PBS buffer, resuspend, and prepare a concentration of 1×10⁻⁶. 9 CFU / mL probiotic suspension;

[0079] S4. Mix the modified peptide solution and probiotic suspension at a volume ratio of 2.5:1, adjust the pH to 7.0, let stand at 37°C for 4 hours, and freeze dry to obtain the modified milk-derived active peptide loaded with probiotics.

[0080] Comparative Example 1

[0081] The difference between this comparative example and Example 3 is that it does not involve grafting polyethylene glycol modification, as detailed below:

[0082] The specific preparation steps of a modified milk-derived bioactive peptide carrying probiotics are as follows:

[0083] S1. Dissolve the decapeptide powder of casein hydrolysate in a 3% CaCl2 solution to prepare a polypeptide solution with a concentration of 5 mg / mL;

[0084] S2. Culture *Lactobacillus plantarum* to the logarithmic growth phase, collect the cells by centrifugation, wash with sterile PBS buffer, resuspend, and prepare a concentration of 1×10⁻⁶. 9 CFU / mL probiotic suspension;

[0085] S3. Mix the polypeptide solution and probiotic suspension at a volume ratio of 2:1, adjust the pH to 7.0, let stand at 37°C for 4 hours, and freeze dry to obtain the probiotic-loaded milk-derived active peptide.

[0086] Comparative Example 2

[0087] The difference between this comparative example and Example 3 is that the amount of polyethylene glycol added is too low. The mass ratio of polyethylene glycol to casein hydrolysate decapeptide is 0.5:1, as detailed below:

[0088] The specific preparation steps of a modified milk-derived bioactive peptide carrying probiotics are as follows:

[0089] S1. Dissolve the casein hydrolysate decapeptide in PBS buffer to prepare a polypeptide solution with a concentration of 10 mg / mL. Then add polyethylene glycol, with a mass ratio of polyethylene glycol to casein hydrolysate decapeptide of 0.5:1. Stir at room temperature for 12 h, dialyze, and freeze dry to obtain modified polypeptide powder.

[0090] S2. Dissolve the modified polypeptide powder in a 3% CaCl2 solution to obtain a modified polypeptide solution with a concentration of 5 mg / mL;

[0091] S3. Culture *Lactobacillus plantarum* to the logarithmic growth phase, collect the cells by centrifugation, wash with sterile PBS buffer, resuspend, and prepare a concentration of 1×10⁻⁶. 9 CFU / mL probiotic suspension;

[0092] S4. Mix the modified peptide solution and probiotic suspension at a volume ratio of 2:1, adjust the pH to 7.0, let stand at 37°C for 4 hours, and freeze dry to obtain the modified milk-derived active peptides loaded with probiotics.

[0093] Comparative Example 3

[0094] The difference between this comparative example and Example 3 is that the amount of polyethylene glycol added is too high. The mass ratio of polyethylene glycol to casein hydrolysate decapeptide is 4:1, as detailed below:

[0095] The specific preparation steps of a modified milk-derived bioactive peptide carrying probiotics are as follows:

[0096] S1. Dissolve the casein hydrolysate decapeptide in PBS buffer to prepare a polypeptide solution with a concentration of 10 mg / mL. Then add polyethylene glycol, with a mass ratio of polyethylene glycol to casein hydrolysate decapeptide of 4:1. Stir at room temperature for 12 h, dialyze, and freeze dry to obtain modified polypeptide powder.

[0097] S2. Dissolve the modified polypeptide powder in a 3% CaCl2 solution to obtain a modified polypeptide solution with a concentration of 5 mg / mL;

[0098] S3. Culture *Lactobacillus plantarum* to the logarithmic growth phase, collect the cells by centrifugation, wash with sterile PBS buffer, resuspend, and prepare a concentration of 1×10⁻⁶. 9 CFU / mL probiotic suspension;

[0099] S4. Mix the modified peptide solution and probiotic suspension at a volume ratio of 2:1, adjust the pH to 7.0, let stand at 37°C for 4 hours, and freeze dry to obtain the modified milk-derived active peptides loaded with probiotics.

[0100] Comparative Example 4

[0101] The difference between this comparative example and Example 3 is that the probiotics were not encapsulated, as detailed below:

[0102] The specific preparation steps of a modified milk-derived bioactive peptide carrying probiotics are as follows:

[0103] S1. Dissolve the casein hydrolysate decapeptide in PBS buffer to prepare a polypeptide solution with a concentration of 10 mg / mL. Then add polyethylene glycol, with a mass ratio of polyethylene glycol to casein hydrolysate decapeptide of 2:1. Stir at room temperature for 12 h, dialyze, and freeze dry to obtain modified polypeptide powder.

[0104] S2. Dissolve the modified peptide powder in a 3% CaCl2 solution to obtain a modified peptide solution with a concentration of 5 mg / mL. Adjust the pH to 7.0, let it stand at 37℃ for 4 hours, and freeze-dry to obtain the modified milk-derived active peptide.

[0105] Comparative Example 5

[0106] 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, at 0.5:1, as detailed below:

[0107] The specific preparation steps of a modified milk-derived bioactive peptide carrying probiotics are as follows:

[0108] S1. Dissolve the casein hydrolysate decapeptide in PBS buffer to prepare a polypeptide solution with a concentration of 10 mg / mL. Then add polyethylene glycol, with a mass ratio of polyethylene glycol to casein hydrolysate decapeptide of 2:1. Stir at room temperature for 12 h, dialyze, and freeze dry to obtain modified polypeptide powder.

[0109] S2. Dissolve the modified polypeptide powder in a 3% CaCl2 solution to obtain a modified polypeptide solution with a concentration of 5 mg / mL;

[0110] S3. Culture *Lactobacillus plantarum* to the logarithmic growth phase, collect the cells by centrifugation, wash with sterile PBS buffer, resuspend, and prepare a concentration of 1×10⁻⁶. 9 CFU / mL probiotic suspension;

[0111] S4. Mix the modified peptide solution and probiotic suspension at a volume ratio of 0.5:1, adjust the pH to 7.0, let stand at 37°C for 4 hours, and freeze dry to obtain the modified milk-derived active peptide loaded with probiotics.

[0112] Comparative Example 6

[0113] The difference between this comparative example and Example 3 is that the volume ratio of the modified peptide solution to the probiotic suspension is too high, at 3:1, as detailed below: A modified milk-derived active peptide carrying probiotics is prepared using the following steps:

[0114] S1. Dissolve the casein hydrolysate decapeptide in PBS buffer to prepare a polypeptide solution with a concentration of 10 mg / mL. Then add polyethylene glycol, with a mass ratio of polyethylene glycol to casein hydrolysate decapeptide of 2:1. Stir at room temperature for 12 h, dialyze, and freeze dry to obtain modified polypeptide powder.

[0115] S2. Dissolve the modified polypeptide powder in a 3% CaCl2 solution to obtain a modified polypeptide solution with a concentration of 5 mg / mL;

[0116] S3. Culture *Lactobacillus plantarum* to the logarithmic growth phase, collect the cells by centrifugation, wash with sterile PBS buffer, resuspend, and prepare a concentration of 1×10⁻⁶. 9 CFU / mL probiotic suspension;

[0117] S4. Mix the modified peptide solution and probiotic suspension at a volume ratio of 3:1, adjust the pH to 7.0, let stand at 37°C for 4 hours, and freeze dry to obtain the modified milk-derived active peptides loaded with probiotics.

[0118] Comparative Example 7

[0119] The difference between this comparative example and Example 3 is that CaCl2 was not added, as detailed below:

[0120] The specific preparation steps of a modified milk-derived bioactive peptide carrying probiotics are as follows:

[0121] S1. Dissolve the casein hydrolysate decapeptide in PBS buffer to prepare a polypeptide solution with a concentration of 10 mg / mL. Then add polyethylene glycol, with a mass ratio of polyethylene glycol to casein hydrolysate decapeptide of 2:1. Stir at room temperature for 12 h, dialyze, and freeze dry to obtain modified polypeptide powder.

[0122] S2. Dissolve the modified polypeptide powder in water to prepare a modified polypeptide solution with a concentration of 5 mg / mL;

[0123] S3. Culture *Lactobacillus plantarum* to the logarithmic growth phase, collect the cells by centrifugation, wash with sterile PBS buffer, resuspend, and prepare a concentration of 1×10⁻⁶. 9 CFU / mL probiotic suspension;

[0124] S4. Mix the modified peptide solution and probiotic suspension at a volume ratio of 2:1, adjust the pH to 7.0, let stand at 37°C for 4 hours, and freeze dry to obtain the modified milk-derived active peptides loaded with probiotics.

[0125] Performance testing:

[0126] (1) Probiotic encapsulation rate

[0127] Table 1. Probiotic encapsulation rate in modified milk-derived bioactive peptides prepared in Examples 1-9 and Comparative Examples 1-7

[0128]

[0129]

[0130] Table 1 shows that the probiotic encapsulation rate in the modified milk-derived active peptides prepared in Examples 1-9 was 80.5-85.6%, with Example 3 showing the highest probiotic encapsulation rate of 85.6%. However, in the comparative examples, the amount of polyethylene glycol added was too low / too high, the volume ratio of the modified peptide solution to the probiotic suspension was too low / too high, and no Ca was added. 2+ The stability and structure of the prepared modified milk-derived bioactive peptides are relatively poor, so the encapsulation rate of probiotics will also decrease accordingly.

[0131] (2) Probiotic release rate

[0132] The modified milk-derived bioactive peptides prepared in the examples and comparative examples were placed in simulated gastric fluid and incubated at 37°C with shaking for 4 hours, with samples taken every 2 hours. The samples treated with gastric fluid were transferred to simulated intestinal fluid and incubated at 37°C with shaking for 4 hours, with samples taken every 2 hours. The released liquid was serially diluted and spread on a culture medium, and the number of viable bacteria released was calculated. The probiotic release rate was calculated using the following formula:

[0133] Probiotic release rate (%) = Number of live bacteria released in the stomach or intestines / Initial number of encapsulated live bacteria × 100%

[0134] Table 2. Probiotic release rates of the modified milk-derived bioactive peptides prepared in Examples 1-9 and Comparative Examples 1-7.

[0135]

[0136]

[0137] As shown in Table 2, the modified milk-derived bioactive peptides prepared in Example 3 exhibited a low probiotic release rate of 14.2% in the stomach, while the highest probiotic release rate of 83.6% in the intestines. This demonstrates the effectiveness of polyethylene glycol-modified peptides and the addition of Ca. 2+ The peptide hydrogel structure formed by post-self-assembly is more stable and can effectively protect probiotics in the stomach, enabling them to be released efficiently in the intestines.

[0138] In summary, the modified milk-derived bioactive peptides prepared in Example 3 were selected for use in the subsequent preparation of sleep-aiding milk beverages.

[0139] Example 10

[0140] The application of modified milk-derived bioactive peptides incorporating probiotics in sleep-aiding milk beverages is as follows:

[0141] (1) Mixing: Mix 1% light cream, 0.1% γ-aminobutyric acid, 0.1% jujube seed powder, 0.05% yam powder, 0.002% edible salt, 0.2% emulsifying stabilizer, 1.8% erythritol, 0.001% sucralose, 0.01% sodium carbonate, and 4% modified milk-derived active peptides carrying probiotics prepared in Example 3. Shear the mixture in 80°C hot water for 15 minutes to fully dissolve it. Then add 68% raw milk and fill with softened water to 85% of the required liquid level. Stir for 10 minutes to mix the liquid evenly. Then add 0.04% edible flavoring and fill to the required volume. Stir for 5 minutes to mix the liquid evenly.

[0142] (2) Homogenization and sterilization: First, homogenize for 15 seconds at a pressure of 20 MPa and a temperature of 60°C; after the liquid cools 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.

[0143] (3) Aseptic filling: Under the condition of filling temperature of 30℃, the liquid is aseptically filled to obtain the sleep aid milk beverage.

[0144] Comparative Example 8

[0145] The difference between this comparative example and Example 10 is that no modified milk-derived active peptides were added, as detailed below:

[0146] (1) Mixing: Mix 1% light cream, 0.1% γ-aminobutyric acid, 0.1% jujube seed powder, 0.05% yam powder, 0.002% edible salt, 0.2% emulsifying stabilizer, 1.8% erythritol, 0.001% sucralose, and 0.01% sodium carbonate. Shear in 80℃ hot water for 15 minutes to fully dissolve the mixture. Add 68% raw milk and fill with softened water to 85% of the required liquid level. Stir for 10 minutes to ensure the mixture is homogeneous. Then add 0.04% edible flavoring and fill to the required volume. Stir for 5 minutes to ensure the mixture is homogeneous.

[0147] (2) Homogenization and sterilization: First, homogenize for 15 seconds at a pressure of 20 MPa and a temperature of 60°C; after the liquid cools 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.

[0148] (3) Aseptic filling: Under the condition of filling temperature of 30℃, the liquid is aseptically filled to obtain the sleep aid milk beverage.

[0149] Comparative Example 9

[0150] The difference between this comparative example and Example 10 is the direct addition of milk-derived active peptides, as detailed below:

[0151] (1) Mixing: Mix 1% light cream, 0.1% γ-aminobutyric acid, 0.1% jujube seed powder, 0.05% yam powder, 0.002% edible salt, 0.2% emulsifying stabilizer, 1.8% erythritol, 0.001% sucralose, 0.01% sodium carbonate, and 4% casein hydrolysate decapeptide. Shear in 80℃ hot water for 15 minutes to fully dissolve the mixture. Add 68% raw milk and fill with softened water to 85% of the required volume. Stir for 10 minutes to ensure the mixture is homogeneous. Then add 0.04% edible flavoring and fill to volume. Stir for 5 minutes to ensure the mixture is homogeneous.

[0152] (2) Homogenization and sterilization: First, homogenize for 15 seconds at a pressure of 20 MPa and a temperature of 60°C; after the liquid cools 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.

[0153] (3) Aseptic filling: Under the condition of filling temperature of 30℃, the liquid is aseptically filled to obtain the sleep aid milk beverage.

[0154] Comparative Example 10

[0155] The difference between this comparative example and Example 10 is that both milk-derived bioactive peptides and probiotics are added directly, as detailed below:

[0156] (1) Mixing: Mix 1% light cream, 0.1% γ-aminobutyric acid, 0.1% jujube seed powder, 0.05% yam powder, 0.002% edible salt, 0.2% emulsifying stabilizer, 1.8% erythritol, 0.001% sucralose, 0.01% sodium carbonate, 3% casein hydrolysate decapeptide, and 1% Lactobacillus plantarum. Shear in 80℃ hot water for 15 minutes to fully dissolve the mixture. Add 68% raw milk and fill with softened water to 85% of the required volume. Stir for 10 minutes to ensure the mixture is homogeneous. Then add 0.04% edible flavoring and fill to volume. Stir for 5 minutes to ensure the mixture is homogeneous.

[0157] (2) Homogenization and sterilization: First, homogenize for 15 seconds at a pressure of 20 MPa and a temperature of 60°C; after the liquid cools 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.

[0158] (3) Aseptic filling: Under the condition of filling temperature of 30℃, the liquid is aseptically filled to obtain the sleep aid milk beverage.

[0159] Comparative Example 11

[0160] The difference between this comparative example and Example 10 is that it uses the modified milk-derived active peptide prepared in Comparative Example 1.

[0161] Comparative Example 12

[0162] The difference between this comparative example and Example 10 is that the modified milk-derived active peptide prepared in Comparative Example 2 is used.

[0163] Comparative Example 13

[0164] The difference between this comparative example and Example 10 is that the modified milk-derived active peptide prepared in Comparative Example 3 is used.

[0165] Comparative Example 14

[0166] The difference between this comparative example and Example 10 is that the modified milk-derived active peptide prepared in Comparative Example 4 is used.

[0167] Comparative Example 15

[0168] The difference between this comparative example and Example 10 is that it uses the modified milk-derived active peptide prepared in Comparative Example 5.

[0169] Comparative Example 16

[0170] The difference between this comparative example and Example 10 is that it uses the modified milk-derived active peptide prepared in Comparative Example 6.

[0171] Comparative Example 17

[0172] The difference between this comparative example and Example 10 is that it uses the modified milk-derived active peptide prepared in Comparative Example 7.

[0173] (1) Evaluation of drinking effects

[0174] The sleep-aid milk beverages prepared in Example 10 and Comparative Examples 8-17 were tasted and investigated by 100 evaluators who drank them continuously for 30 days to determine their sleep-improvement effects. The results are shown in Table 3.

[0175] Table 3. Evaluation of the drinking effects of the sleep-aiding milk beverages prepared in Example 10 and Comparative Examples 8-17

[0176]

[0177] 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. This indicates that the addition of modified milk-derived active peptides with encapsulated probiotics is more helpful in improving sleep.

[0178] (2) Evaluation of sleep-aiding effect

[0179] 1. Animal grouping and establishment of an insomnia mouse model

[0180] All mice (weighing 20±3g) were free of specific pathogens. Healthy female SPF-grade Kunming mice were provided by the Shanghai Laboratory Animal Center and fed a standard pelleted rodent diet and purified water. All procedures involving the mice complied with the guidelines provided by the Shanghai Laboratory Animal Care and Animal Experimentation Center.

[0181] A total of 96 mice (8 mice / group) were used, and 88 mice were randomly selected to establish an insomnia model using a modified horizontal rotating disc sleep deprivation method. A sleep deprivation box measuring 110cm long, 60cm wide, and 40cm high was constructed using an acrylic plastic board. Fifteen cylindrical platforms, each 8cm high and 6.5cm in diameter, were fixed to the bottom of the box with a standard longitudinal and lateral distance of 10cm. Water at 23-25℃ was poured to a depth of 1cm below the platforms to maintain the water temperature. After 7 days of acclimatization, the mice were trained to stand. They were placed on the cylindrical platforms, and when they were about to fall asleep, they were startled awake by their muscles relaxing and their heads drooping into the water or falling in, thus forcing them to remain awake and standing. During the 2-week modeling period, the mice rested twice a day, morning and evening, for 30 minutes each time. Behavioral changes, including their mental state and diet, were recorded every two days.

[0182] Eighty-eight insomnia model mice were randomly divided into 11 groups: Example 10, Comparative Example 8, Comparative Example 9, Comparative Example 10, Comparative Example 11, Comparative Example 12, Comparative Example 13, Comparative Example 14, Comparative Example 15, Comparative Example 16, and Comparative Example 17. The remaining eight mice that did not undergo modeling served as the control group. At the beginning of the second week of standing training, mice were administered a sleep-aiding milk beverage via gavage, while the control group mice were given the same volume of purified water. Twelve hours after the last gavage administration (four weeks of sleep-aiding milk beverage intervention), the mice were euthanized by an overdose of sodium pentobarbital.

[0183] 2. Sleep Improvement Test

[0184] All tests were conducted in a quiet environment and were carried out the day before the end of the experiment, as follows:

[0185] a. Pentobarbital sodium-induced sleep duration test: In the sleep test, sleep and wakefulness were determined by the disappearance of the righting reflex, with the disappearance of the righting reflex for more than 30 seconds considered as sleep. Mice in each group were administered pentobarbital sodium (49 mg / kg bw) intraperitoneally 30 minutes after gavage. The duration of sleep was recorded as the time interval from the disappearance of the righting reflex to its recovery.

[0186] Depend on Figure 1It can be seen that, compared with the control group, the symptoms of insomnia mice in Group 10 of the present invention were relieved by gavage and the sleep time was significantly prolonged. In contrast, the sleep time was the shortest in Group 8 of the comparative example, which did not contain modified milk-derived active peptides. In Group 14 of the comparative example, the lack of probiotics did not result in a significant effect on improving sleep.

[0187] b. Pentobarbital sodium-induced sleep latency test: 30 minutes after drug intervention, each mouse was intraperitoneally injected with pentobarbital sodium at a dose of 320 mg / kg bw. Sleep latency was recorded as the time interval from pentobarbital sodium injection to the disappearance of the righting reflex.

[0188] Depend on Figure 2 It can be seen that, compared with the control group, the sleep latency of the insomnia mice in Group 10 of Example 10 was significantly shortened, and the insomnia mice were more likely to fall asleep; while the sleep latency of Group 8 of the comparative example, which did not add modified milk-derived active peptides, was prolonged; and the sleep latency of Group 14 of the comparative example, which lacked probiotics, was also relatively long, and the insomnia mice still had relatively severe sleep onset difficulties.

[0189] 3. Determination of 5-hydroxytryptamine (5-HT) content in the brain: After the mice were sacrificed, the whole brain was immediately removed by craniotomy under ice bath. The brain was separated on an ice-cold petri dish, and the blood was rinsed with 0.9% ice-cold sodium chloride solution. After drying with filter paper, the brain was weighed, and 9 times the volume of 0.9% ice-cold NaCl solution was added. The mixture was homogenized at 4°C, and the homogenate was transferred to a 2 mL centrifuge tube. The mixture was centrifuged at 3000 r / min for 15 min at 4°C. The supernatant was collected, and the 5-HT content was determined according to the relevant operating steps in the kit instructions (purchased from Wuhan Pure Biotechnology Co., Ltd.).

[0190] Depend on Figure 3 It can be seen that, compared with the control group, the 5-hydroxytryptamine content of the insomnia mice in Example 10 of this invention was significantly increased, and was also higher than that of the control group. The modified milk-derived active peptides prepared in this invention also contain probiotics. The porous structure of the polypeptide hydrogel can play a sustained-release role for the probiotics, so that the delivered probiotics can regulate the production of 5-hydroxytryptamine in the intestinal environment and enter the nerve cells through the blood. They can bind to the 5-hydroxytryptamine receptors between neurons and play a role in regulating mood, thereby playing a role in preventing sleep disorders, improving sleep quality, and relieving anxiety.

[0191] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any person skilled in the art can make many possible variations and modifications to the technical solutions of the present invention, or modify them into equivalent embodiments, without departing from the spirit and technical essence of the present invention. Therefore, any simple modifications, equivalent substitutions, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention, without departing from the content of the technical solutions of the present invention, shall still fall within the scope of protection of the present invention.

Claims

1. A method for preparing modified milk-derived bioactive peptides loaded with probiotics, characterized in that: The modified milk-derived active peptides carrying probiotics are obtained by self-assembling the modified milk-derived active peptides into a hydrogel and then encapsulating the probiotics. The modified milk-derived active peptide was obtained by introducing polyethylene glycol into the casein hydrolysate decapeptide YLGYLEQLLR. 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 h, dialyze, and freeze dry to obtain modified polypeptide powder; the mass ratio of polyethylene glycol to casein hydrolysate decapeptide is (1-3):

1. S2. Dissolve the modified polypeptide powder in a 2-5% CaCl2 solution to obtain a modified polypeptide solution; S3. The probiotics are cultured to the logarithmic phase, centrifuged to collect the bacterial cells, washed with sterile PBS buffer, and resuspended to obtain a probiotic suspension; the probiotics are Lactobacillus plantarum. S4. Mix the modified peptide solution and probiotic suspension, adjust the pH to 6.5-7.5, let stand at 30-37℃ for 2-5 hours, and freeze dry to obtain the modified milk-derived active peptides carrying probiotics.

2. The method for preparing modified milk-derived bioactive peptides carrying probiotics according to claim 1, characterized in that, In step S1, the concentration of casein hydrolysate decapeptide is 10-15 mg / mL.

3. The method for preparing modified milk-derived bioactive peptides carrying probiotics according to claim 1, characterized in that: The concentration of the modified polypeptide solution in step S2 is 2-10 mg / mL.

4. The method for preparing modified milk-derived bioactive peptides carrying probiotics according to claim 1, characterized in that: The concentration of the probiotic suspension is 1×10⁻⁶. 8 -1×10 9 CFU / mL.

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

1.

6. The application of the modified milk-derived bioactive peptides carrying probiotics prepared by the preparation method according to any one of claims 2-5 in the preparation of sleep-aiding milk beverages.

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