Casein hydrolysate and application thereof in preparation of medicine for preventing or treating nerve aging

Abstract

The invention relates to casein peptide hydrolysate and application thereof in preparation of drugs for preventing or treating nerve aging, and belongs to the technical field of biological medicines. The casein hydrolysate is obtained by fermenting a casein solution with the probiotic bifidobacterium longum BNCC 185354, and the obtained casein hydrolysate has superoxide anion free radical scavenging capacity, can prolong the telomere length of the hippocampus of a subacute aging model mouse and relieve DNA oxidative damage of the hippocampus of the mouse. The brand-new casein peptide with both brain-accessible activity and biological activity is further screened from casein hydrolysate, free radicals can be removed, nerve cell inflammation can be eliminated, and therefore nerve aging is delayed. The casein peptide and the casein hydrolysate provided by the invention can be applied to preparation of drugs for preventing and / or treating nerve aging, and have good application prospects.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to casein hydrolysate and its application in the preparation of drugs for the prevention or treatment of neurosenescence. Background Technology

[0002] With the accelerating aging process and increasing average lifespan, aging and related diseases are the biggest obstacles to achieving healthy aging. The essence of aging is cellular senescence, which manifests as permanent cell cycle arrest, impaired cell function and intercellular communication, neuroinflammation, and damage to other neighboring cells.

[0003] Oxidative stress is a crucial pathological mechanism in neuroaging. Oxidative stress refers to the imbalance between oxidants and antioxidants in the body, leading to the excessive accumulation of reactive oxygen species (ROS) and reactive nitrogen species (RNS). This excessive accumulation damages cellular lipids, proteins, and DNA, thereby triggering cellular dysfunction and cell death. Neuroinflammation is also considered a major cause of neuroaging. Neuroinflammation refers to the inflammatory response in the central nervous system, primarily caused by the activation of microglia and astrocytes. The activation of these cells releases pro-inflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6), thereby damaging neurons. Given the significant role of antioxidants and anti-inflammatories in neuroaging, reducing oxidative stress and neuroinflammation could lead to the development of more effective therapeutic strategies to protect neurons, delay telomere wear, and prevent neuroaging.

[0004] Current interventions primarily focus on improving symptoms in the clinical stage, with few effective interventions targeting the early stages of the disease. Treatment options are also quite limited, mainly including cholinesterase inhibitors and N-methyl-D-aspartate receptor antagonists. These drugs primarily provide symptomatic treatment and do not slow disease progression; moreover, they may cause a range of side effects. Therefore, developing safe and effective interventions to address neurosensitivity occurring in the early stages of the disease is of great significance. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention utilizes the probiotic Bifidobacterium longum BNCC 185354 to ferment casein solution to obtain casein hydrolysate, verifies the potential of casein hydrolysate in preventing or treating neuroaging, and screens out novel casein peptides with brain-penetrating and biological activity.

[0006] The first object of the present invention is to provide a casein hydrolysate obtained by hydrolyzing casein with Bifidobacterium longum BNCC 185354.

[0007] Furthermore, the casein hydrolysate contains one or more casein peptides with the following amino acid sequence:

[0008] (1) SEQ ID NO.1;

[0009] (2) SEQ ID NO.3;

[0010] (3) SEQ ID NO.6;

[0011] (4) SEQ ID NO.7;

[0012] (5) SEQ ID NO.8.

[0013] A second objective of this invention is to provide a method for preparing the above-mentioned casein hydrolysate, comprising the following steps:

[0014] Step S1: Prepare a casein solution, wherein the casein solution is obtained by mixing casein and an inorganic base in water;

[0015] Step S2: Mix the activated Bifidobacterium longum BNCC 185354 with the casein solution, ferment, centrifuge and collect the supernatant to obtain the casein hydrolysate.

[0016] Further, in step S1, the mass ratio of casein to water is 2-3:97-98, and the mass ratio of inorganic alkali to water is 4-5:95-100.

[0017] Further, in step S2, the activated Bifidobacterium longum BNCC 185354 is added to the Bifidobacterium longum liquid culture medium and cultured in an anaerobic incubator at 35-40℃ for 30-40 h.

[0018] Further, in step S2, the amount of Bifidobacterium longum BNCC 185354 added is (3-7) × 10⁻⁶. 8 CFU / mL.

[0019] Furthermore, in step S2, the fermentation conditions are as follows: fermentation in an anaerobic incubator at 35-40°C for 70-75 hours.

[0020] Further, in step S2, the centrifugation conditions are: centrifugation at 3500-4500 rpm / min for 5-15 minutes at 0-5℃.

[0021] In one embodiment of the present invention, the inorganic base is sodium hydroxide (NaOH).

[0022] In one embodiment of the present invention, the sodium hydroxide is a 0.5% aqueous solution.

[0023] A third object of the present invention is to provide a casein peptide having an amino acid sequence as shown in SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO.8.

[0024] Furthermore, the amino terminus and / or carboxyl terminus of the casein peptide are modified.

[0025] Furthermore, the modification is one or more of amidation, sulfation, acetylation, and fatty acidation.

[0026] A fourth object of the present invention is to provide a gene sequence encoding the above-mentioned casein peptide.

[0027] A fifth object of the present invention is to provide the use of the above-mentioned casein hydrolysate or casein peptide in the preparation of a medicament for preventing and / or treating neurosenescence, said medicament having at least one of the following functions:

[0028] (1) Clear reactive oxygen free radicals in nerve cells;

[0029] (2) Extend the telomere length in the hippocampus;

[0030] (3) Alleviate oxidative damage to DNA in the hippocampus;

[0031] (4) Reduce the concentration of malondialdehyde in nerve cells;

[0032] (5) Reduce the concentration of nitric oxide in nerve cells;

[0033] (6) Reduce the concentration of interleukin-1β in nerve cells.

[0034] A sixth object of the present invention is to provide a medicament for preventing and / or treating neurosenescence, the medicament comprising the above-mentioned casein hydrolysate or the above-mentioned casein peptide, the medicament having at least one of the following functions:

[0035] (1) Clear reactive oxygen free radicals in nerve cells;

[0036] (2) Extend the telomere length in the hippocampus;

[0037] (3) Alleviate oxidative damage to DNA in the hippocampus;

[0038] (4) Reduce the concentration of malondialdehyde in nerve cells;

[0039] (5) Reduce the concentration of nitric oxide in nerve cells;

[0040] (6) Reduce the concentration of interleukin-1β in nerve cells.

[0041] The beneficial effects of this invention are:

[0042] (1) The present invention uses the probiotic Bifidobacterium longum BNCC 185354 to ferment and prepare casein hydrolysate, which has the advantages of better safety and lower cost. The casein hydrolysate prepared has the effect of improving and delaying neurosensitivity and can be used as an effective intervention for early symptoms of neurosensitivity. Moreover, the preparation method is simple, low cost, easy to scale up production, and avoids unknown health risks that may be caused by the introduction of exogenous substances.

[0043] (2) The present invention screened novel casein peptides with amino acid sequences such as SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO.8 from casein hydrolysate. These peptides have brain-penetrating and biological activities, and also have the effect of improving and delaying neurosenescence. Furthermore, the dual antioxidant and anti-inflammatory activities were verified by constructing a mouse model, providing novel drug candidates for the prevention and / or treatment of neurosenescence. Attached Figure Description

[0044] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein:

[0045] Figure 1 The qualitative and quantitative detection results of casein fermentation products in Example 2 of the present invention are shown, where A is the peptide concentration and B is the degree of protein hydrolysis.

[0046] Figure 2 This is a liquid chromatogram of the casein fermentation product in Example 2 of the present invention;

[0047] Figure 3 The results of superoxide anion free radical scavenging of casein fermentation products in Example 3 of this invention are shown.

[0048] Figure 4 The results of the detection of the effect of casein hydrolysate on the relative telomere length of rat hippocampus in Example 4 of the present invention;

[0049] Figure 5 The results of the detection of the effect of casein hydrolysate on DNA oxidative damage in the rat hippocampus in Example 5 of the present invention;

[0050] Figure 6 This is the brain-accessible activity screening result of casein peptides in Example 6 of the present invention;

[0051] Figure 7 The results of the detection of the effect of casein peptide on intracellular reactive oxygen species level in Example 7 of the present invention;

[0052] Figure 8The results of the detection of the effect of casein peptide on intracellular malondialdehyde levels in Example 7 of the present invention;

[0053] Figure 9 The results of the detection of the effect of casein peptide on intracellular nitric oxide concentration in Example 7 of the present invention;

[0054] Figure 10 The results of the detection of the effect of casein peptide on the concentration of interleukin-1β in cells in Example 7 of the present invention;

[0055] Figure 11 The results show the effect of casein peptide on the relative telomere length of neural stem cells in Example 8 of this invention. Detailed Implementation

[0056] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0057] The raw materials and equipment used in this invention are all known products, obtained by purchasing commercially available products.

[0058] The "room temperature" referred to in this invention is 25±5℃.

[0059] The biuret reagent formulation used in this invention is as follows:

[0060] Dissolve 1.5 g of copper sulfate pentahydrate and 6.0 g of sodium potassium tartrate tetrahydrate in ultrapure water, then add 1.0 g of potassium iodide. While stirring, add 300 mL of 10% (w / v) NaOH solution and bring the volume to 1000 mL. Store in a brown bottle at room temperature.

[0061] The culture medium used in this invention is as follows:

[0062] BBL medium: purchased from Haibo Biotechnology, product number HB87777, with the following composition: peptone 15.0 g / L, glucose 20.0 g / L, yeast extract 2.0 g / L, soluble starch 0.5 g / L, sodium chloride 5.0 g / L, L-cysteine ​​0.5 g / L, tomato extract 2.0 g / L, Tween 80 1.0 g / L.

[0063] BV2 cell culture medium: purchased from Pronosai, catalog number CM-0493A; composition as follows: DMEM basal medium + 10% fetal bovine serum + 1% penicillin-dextrose antibody.

[0064] Complete culture medium for rat hippocampal neural stem cells: DMEM / F12 medium supplemented with 10 mL of B27, 1% penicillin-streptomycin, basic fibroblast growth factor at a final concentration of 20 ng / mL, and epidermal growth factor at a final concentration of 20 ng / mL.

[0065] Example 1: Preparation of casein hydrolysate by fermentation of Bifidobacterium longum BNCC 185354

[0066] (1) Bacterial cell treatment

[0067] After resuscitation, *Bifidobacterium longum* BNCC 185354 was reactivated by subculturing twice. Then, it was added at a ratio of 1%-2% to sterile *Bifidobacterium longum* liquid culture medium (BBL), mixed thoroughly, and incubated in a 37℃ anaerobic incubator for 36 h for bacterial amplification. After incubation, the bacterial culture was concentrated by centrifugation, and plate counting was performed according to national standard methods. The plate counts were then transferred to sterile 50 mL centrifuge tubes, ensuring each tube contained 5 × 10⁻⁶ bacteria. 8 CFU / mL, centrifuged at 3000 rpm / min for 10 min at 4℃, then the supernatant was discarded. Then, 3 volumes of PBS were added for washing, and centrifuged again at 3000 rpm / min for 10 min at 4℃. The PBS was then discarded and the solution was ready for use.

[0068] (2) Preparation of casein solution

[0069] Weigh casein solids into a beaker at a ratio of 2.5 g casein (Sigma, CAS: 9000-71-9, milk source) / 100 mL ultrapure water. Add 0.5% NaOH solution at 4% (v / v) and stir thoroughly. Then add 96% (v / v) ultrapure water, seal with sealing film, and stir magnetically at room temperature. After the casein clumps are completely dissolved, the pH is measured to be between 7.2 and 7.3. Sterilize by heating at 90℃ for 15 min, and then cool to room temperature to obtain a casein solution.

[0070] (3) Preparation of protein hydrolysate

[0071] First, take a small amount of casein solution to resuspend the bacterial precipitate obtained in step (1), then add the remaining casein solution and mix well to maintain the bacterial count in the fermentation system at 5 × 10⁻⁶. 8 CFU / mL; then screw on the centrifuge tube cap, seal with sealing film, and place in an anaerobic incubator at 37℃ for 72 h of fermentation. After fermentation, centrifuge the fermentation system at 4℃ and 3900 rpm / min for 10 min. At this time, the supernatant is transparent. Filter with a 0.22 μm membrane to remove bacteria to obtain the fermented casein hydrolysate.

[0072] Example 2: Qualitative and quantitative testing of casein hydrolysates

[0073] (1) Degree of protein hydrolysis

[0074] The phthalaldehyde method was used to determine the free amino acid content in casein hydrolysates, thereby characterizing the degree of protein hydrolysis. The specific steps are as follows: First, prepare the phthalaldehyde (OPA) reagent: Dissolve 7.620 g of sodium tetraborate and 200 mg of sodium dodecyl sulfate (SDS) in 150 mL of deionized water to obtain a mixed solution; dissolve 160 mg of phthalaldehyde (OPA) in 4 mL of ethanol, then add the OPA ethanol solution to the above mixed solution, and then add 176 mg of dithiothreitol. Stir magnetically until completely dissolved, and finally dilute to 200 mL with deionized water. Simultaneously, prepare a serine (0.1 mg / mL serine) standard solution. Mix 3 mL of the OPA reagent with 400 μL of the serine standard solution or sample solution, shake well, and measure the absorbance at 340 nm. Calculate the degree of protein hydrolysis using a standard curve.

[0075] (2) Polypeptide concentration

[0076] The peptide concentration in protein hydrolysates was determined using the biuret reaction method. The specific steps were as follows: First, Gly-Gly-Tyr-Arg tetrapeptide standard solutions with concentration gradients of 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, and 1.8 mg / mL were prepared sequentially using 5% (v / v) trichloroacetic acid (TCA). Then, 6.0 mL of each standard solution was taken, and 4.0 mL of biuret reagent was added. The mixture was vortexed and allowed to stand for 10 min. The mixture was then centrifuged at 2000 rpm for 10 min. The supernatant was collected and the OD value was measured at 540 nm. A standard curve was constructed with peptide concentration as the x-axis (mg / mL) and OD value as the y-axis. Take 2.5 mL of sample solution, add 2.5 mL of 10% (v / v) trichloroacetic acid solution, mix thoroughly on a vortex mixer, let stand for 10 min, then centrifuge at 4000 rpm / min for 15 min. Transfer the supernatant to a 50 mL volumetric flask and dilute to volume with 5% TCA, then shake well. Take 6.0 mL of the above solution and place it in another test tube, add 4.0 mL of biuret reagent (sample solution:biuret reagent = 3:2, v / v), mix thoroughly on a vortex mixer, let stand for 10 min, centrifuge at 2000 rpm / min for 10 min, take the supernatant and measure the OD value at a wavelength of 540 nm. Determine the peptide concentration C (mg / mL) in the sample solution by referring to the standard curve, and then determine the peptide content in the sample.

[0077] The results are as follows Figure 1As shown in A and B, the peptide concentration and degree of protein hydrolysis in the fermentation product were 11.73±1.96 mg / mL and 16.67±2.68 mg L-ser / mL, respectively. Figure 2 The liquid chromatogram of the fermentation product is shown. Compared with the unfermented casein control, the number, height and area of ​​the chromatographic peaks are significantly increased, revealing that a large number of casein peptides are released during the fermentation process, mostly hydrophilic peptides.

[0078] Example 3: Determination of the superoxide anion free radical scavenging ability of casein hydrolysate

[0079] The superoxide anion radical scavenging activity assay kit (Maclean) was used to detect the superoxide anion radical scavenging rate of casein hydrolysate prepared by fermentation of Bifidobacterium longum BNCC 185354 using an enzyme-linked immunosorbent assay (ELISA) according to the instructions.

[0080] The results are as follows Figure 3 As shown, compared with the control group, the chemical antioxidant activity of the fermentation product was significantly increased to 43.05±8.97%.

[0081] Example 4: Effect of casein hydrolysate on relative telomere length in the hippocampus of D-galactose-induced SD rats

[0082] The D-galactose-induced SD rat model is a widely used subacute aging model for studying neuroaging. This model induces the aging process in rats through subcutaneous or intraperitoneal injection of D-galactose, mimicking age-related phenotypes in humans.

[0083] After one week of acclimatization, 24 three-month-old male SD rats were randomly divided into four groups: healthy control group (C), D-galactose model group (MC), unfermented casein intervention group (UC), and Bifidobacterium longum fermented casein hydrolysate group (LB), with six rats in each group. Except for the healthy control group, which received daily intraperitoneal injections of an equal volume of sterile saline, all other animals received daily intraperitoneal injections of 100 mg / kg bw D-galactose (dissolved in saline at a concentration of 10 mg / mL). The healthy control group (C) and the model group (MC) were administered ultrapure water (3 mL / rat) by gavage. The unfermented casein group (UC) was administered an equal volume of unfermented casein solution (3 mL / rat) by gavage. The Bifidobacterium longum fermented casein hydrolysate group (LB) was administered Bifidobacterium longum fermented casein hydrolysate by gavage (3 mL / rat). Modeling and intervention were performed simultaneously, with daily gavage and intraperitoneal injections. After 12 weeks of continuous intervention, brain tissue samples were collected for relative telomere length measurement.

[0084] DNA was extracted from rat hippocampal tissue using a kit to detect relative telomere length, with AT1 as an internal control. Primer sequences are as follows:

[0085] 36B4_F: 5'-CAGCAAGTGGGAAGGTGTAATCC-3';

[0086] 36B4_R: 5'-CCCATTCTATCATCAACGGGTACAA-3';

[0087] AT1-F: 5'-ACGTGTTCCAGCATCGACCCTACC-3';

[0088] AT1-R: 5'-AGAATGATAAGGAAAGGGAAGAAGCCC-3'.

[0089] Amplification was performed using Takara RR820A SYBR® Premix Ex Taq™ II (Takara) reagent, and quantitative fluorescence analysis was performed using a Light Cycler96 Real-time PCR instrument. AT1 was used as an internal control, and relative quantification was performed using the 2-ΔΔCt method.

[0090] The results are as follows Figure 4 As shown, the relative telomere length in the hippocampus of the D-galactose-induced model group animals was shorter than that of the normal control group, and the intervention of unfermented casein did not improve the relative telomere length in the hippocampus. However, the casein hydrolysate prepared by fermentation of Bifidobacterium longum BNCC 185354 effectively protected the telomere shortening under D-galactose intervention, and the differences were statistically significant compared with the model group or the unfermented casein control group.

[0091] The above results indicate that the casein hydrolysate prepared by fermentation of *Bifidobacterium longum* BNCC 185354 can protect against D-galactose-induced shortening of relative telomere length in the hippocampus of SD rats. As is known to those skilled in the art, telomere shortening is an important marker of cellular senescence. The fact that the casein hydrolysate prepared by fermentation of *Bifidobacterium longum* BNCC 185354 can protect against D-galactose-induced shortening of relative telomere length in the hippocampus of SD rats suggests that it can delay the aging of nerve cells and thus protect the overall function of the nervous system.

[0092] Example 5: Effects of casein hydrolysate on D-galactose-induced DNA oxidative damage in the hippocampus of SD rats

[0093] The 8-OHdG content in the rat hippocampus was quantified using a rat 8-hydroxydeoxyguanosine (8-OHdG) ELISA kit. A certain amount of hippocampal tissue was weighed, and 10 mg of tissue was added to 90 μL of physiological saline. The tissue was homogenized using a homogenizer until no tissue clumps were observed in the homogenate tube. The homogenate was then centrifuged at 3000 rpm / min for 10 min at 4°C, and the supernatant was collected for testing. The sample was diluted 5 times according to the manufacturer's instructions before testing. Subsequent steps were performed according to the manufacturer's instructions. The absorbance was measured at 450 nm using an ELISA reader, and the sample concentration was calculated based on the standard curve and converted to the content in the hippocampal tissue (unit: ng / mg).

[0094] The results are as follows Figure 5 As shown, the 8-OHdG level in the hippocampus of the model group animals was significantly increased, indicating that D-galactose caused DNA oxidative damage in the hippocampus of SD rats. The casein hydrolysate prepared by fermentation of Bifidobacterium longum BNCC 185354 effectively alleviated the above-mentioned damage effect. The 8-OHdG level of unfermented casein after intervention was not statistically different from that of the model group.

[0095] The above results indicate that the casein hydrolysate prepared by fermentation of *Bifidobacterium longum* BNCC 185354 can effectively alleviate D-galactose-induced DNA oxidative damage in the hippocampus of SD rats. As is known to those skilled in the art, D-galactose-induced oxidative damage disrupts the normal function of hippocampal neurons, thereby affecting cognitive function. Furthermore, oxidative damage often triggers neuroinflammation, which is one of the important mechanisms underlying its development. The casein hydrolysate prepared by fermentation of *Bifidobacterium longum* BNCC 185354 of this invention helps protect hippocampal neurons by reducing hippocampal DNA oxidative damage and mitigating neuroinflammatory responses, thereby improving the learning and memory abilities of rats.

[0096] Example 6: Screening of casein peptides in casein hydrolysate prepared by fermentation of Bifidobacterium longum BNCC 185354

[0097] (1) Screening for brain activity

[0098] Referring to the method in Example 1 of the published patent CN118702799A, "A Casein-Derived Active Peptide That Can Delay Telomere Wear and Its Application" and "A Drug Containing the Active Peptide," brain-penetrating active casein peptides were screened. After preparing fermented casein hydrolysate, the three steps of digestion, absorption, and transport were simulated in vitro in sequence: (1) the human digestion process was simulated using the INFOGEST standard method; (2) the simulated digestion product obtained in a transwell chamber was used to simulate the human intestinal absorption process using a human colon cancer cell (Caco-2) barrier model; (3) the simulated absorption product obtained in a transwell chamber was used to simulate the human blood-brain barrier penetration process using an immortalized human brain microvascular endothelial cell (hCMEC / D3) barrier model.

[0099] After collecting and lyophilizing the products from each stage, Nano-HPLC-MS / MS analysis was performed to identify the peptides contained in each stage. The intersection of the products from each stage was used as the casein peptides released by Bifidobacterium longum BNCC 185354 during fermentation that can resist degradation by endogenous enzymes and have the potential to cross the blood-brain barrier.

[0100] The results are as follows Figure 6 As shown, this invention identified 1971, 663, 276, and 208 casein peptides, respectively, from casein hydrolysate (A), its simulated digestion product (B), simulated absorption product (C), and simulated blood-brain barrier transport product (D) fermented by *Bifidobacterium longum*. The intersection of these identified peptides contained 68 casein peptides, which are the casein peptides with brain-penetrating potential identified in this invention from *Bifidobacterium longum* fermented casein hydrolysate. These 68 casein peptides mainly originated from β-casein and α-S1 casein, primarily consisting of heptapeptides, nonapeptides, and undecapeptides. In subsequent analysis, sequences with extremely low content that could not be quantified were excluded, and sequences with variable modifications were counted as the same sequence as the original sequence. These casein peptides were then further screened.

[0101] (2) Bioactivity screening

[0102] The bioactivity of casein peptides screened in step (1) was predicted using PeptideRanker (http: / / distilldeep.ucd.ie / PeptideRanker / ), and the top ten casein peptides predicted by PeptideRanker were obtained.

[0103] The results are shown in Table 1. The casein peptide with the amino acid sequence SEQ ID NO.1 had the highest Ranker score, indicating that this sequence is the most likely casein peptide with biological activity potential. Furthermore, no reports on this sequence have been found, suggesting that this casein peptide is a newly discovered sequence.

[0104] Table 1. Basic information on casein peptides with brain penetration potential and their PeptideRanker prediction scores.

[0105] serial number polypeptide sequence Molecular weight (Da) Retention time (s) Predicted bioactivity value SEQ ID NO.1 QRFLEPYF 1098.55 50.82 0.77 SEQ ID NO.2 WMHQPHQP 1075.47 9.39 0.74 SEQ ID NO.3 FSDIPNP 788.37 37.29 0.73 SEQ ID NO.4 FVAPFPE 805.40 52.81 0.66 SEQ ID NO.5 GPIVLNPWDQVKR 1520.85 49.97 0.66 SEQ ID NO.6 HQPLPPT 788.42 14.48 0.61 SEQ ID NO.7 WMHQPH 834.36 9.74 0.59 SEQ ID NO.8 GPIVLNPWDQVK 1364.75 52.52 0.57 SEQ ID NO.9 QGPIVLNPWDQVK 1492.80 52.58 0.57 SEQ ID NO.10 YQGPIVLNPWDQVKR 1811.97 51.31 0.56

[0106] Example 7: Validation of the bioactivity of screened casein peptides in lipopolysaccharide-induced BV2 cells

[0107] This invention verifies the bioactivity of casein peptide (SEQ ID NO. 1) in lipopolysaccharide-induced BV2 cells (mouse microglia). The peptide was synthesized by Sangon Biotech (Shanghai) Co., Ltd. using a solid-phase synthesis method, with a purity >99%.

[0108] (1) Reactive oxygen species (ROS) test

[0109] BV2 cells were loaded at 5 × 10 4 The cells were seeded in 24-well plates at a density of cells / well, and the intervention began after 24 h of incubation. The treatment measures for each group were as follows: Control group: no peptides or lipopolysaccharide added, with an equal volume of culture medium added; Model group: no peptides added, only lipopolysaccharide added to a final concentration of 1.0 μg / mL; Positive control group: intervention with lipopolysaccharide to a final concentration of 1.0 μg / mL and curcumin to a final concentration of 5 μM; Casein peptide (SEQ ID NO.1) intervention group: lipopolysaccharide to a final concentration of 1.0 μg / mL and casein peptide (SEQ ID NO.1) at different concentrations (0.01 μg / mL, 0.1 μg / mL, 1.0 μg / mL). All groups were intervened for 24 h. ROS levels were detected using an enzyme-linked immunosorbent assay (ELISA) kit (Beyotime). Specifically, DCFH-DA was diluted 1:1000 with serum-free DMEM to a final concentration of 10 μmol / L. After removing the cell culture medium from the 24-well plate, diluted DCFH-DA was added, and the cells were incubated at 37ºC for 20 min. After scraping off the cells, they were washed three times with serum-free cell culture medium. The cells were then mixed and divided into two aliquots. One aliquot was used to measure the fluorescence intensity at an excitation wavelength of 488 nm and an emission wavelength of 525 nm using a fluorescence microplate reader. The other aliquot was used to measure the absorbance at 450 nm using the CCK-8 assay. The ratio of fluorescence intensity to absorbance at 450 nm was calculated and corrected using the control group value to obtain the relative ROS value.

[0110] (2) MDA level detection

[0111] BV2 cells were loaded at 3 × 10 5Cells were seeded in 6-well plates at a density of cells / well and cultured for 24 h before intervention. The treatments for each group were as follows: Control group: no peptides or lipopolysaccharide added, with an equal volume of culture medium added; Model group: no peptides added, only lipopolysaccharide added to a final concentration of 1.0 μg / mL; Positive control group: lipopolysaccharide and curcumin at a final concentration of 5 μM were added; Casein peptide (SEQ ID NO.1) intervention group: lipopolysaccharide at a final concentration of 1.0 μg / mL and casein peptide (SEQ ID NO.1) at different concentrations (0.01 μg / mL, 0.1 μg / mL, 1.0 μg / mL) were added. All groups were intervened for 24 h. After intervention, cells were scraped off with a cell scraper, lysed, and protein quantification was performed using a BCA kit (Beyotime). MDA levels were detected using an MDA detection kit (Beyotime). The experimental procedures were performed strictly according to the manufacturer's instructions.

[0112] (3) Detection of NO production and IL-1β expression

[0113] BV2 cells were fed at a rate of 1×10 4 Cells were seeded at a density of cells / well in 96-well plates. After 24 h of cell growth, the treatment measures for each group were as follows: Control group: no peptides or lipopolysaccharide added, and equal volume of culture medium added; Model group: no peptides added, only lipopolysaccharide added to a final concentration of 1.0 μg / mL; Positive control group: lipopolysaccharide and curcumin at a final concentration of 5 μM were added; Casein peptide (SEQ ID NO.1) intervention group: lipopolysaccharide at a final concentration of 1.0 μg / mL and casein peptide (SEQ ID NO.1) at different concentrations (0.01 μg / mL, 0.1 μg / mL, 1.0 μg / mL) were added. After 24 h of intervention, cell supernatant was collected, and NO production was detected using the Griess method. IL-1β levels were detected using an ELISA kit.

[0114] like Figure 7 As shown, the reactive oxygen species (ROS) in the model group significantly increased after lipopolysaccharide (LPS) intervention, while the ROS in the positive control group decreased, and the degree of decrease was statistically significant compared with the model group. Intervention with 0.1 μg / mL and 1.0 μg / mL casein peptide (SEQ ID NO. 1) also significantly reduced ROS, with statistically significant differences compared with the model group. 1.0 μg / mL casein peptide (SEQ ID NO. 1) possesses ROS scavenging capacity nearly equivalent to that of 5 μM curcumin.

[0115] (2) Results of malondialdehyde (MDA) test

[0116] MDA test results are as follows Figure 8As shown, the MDA level in cells of the model group increased significantly after lipopolysaccharide induction. The MDA level decreased most significantly in the intervention groups of 0.1 μg / mL or 1.0 μg / mL casein peptide (SEQ ID NO.1), and the difference was statistically significant compared with the model group.

[0117] (3) Results of NO production and IL-1β expression detection

[0118] NO test results are as follows Figure 9 As shown, compared with the uninterrupted control group, the NO production in the model group was significantly increased, and the concentration of nitric oxide was significantly reduced by 0.1 μg / mL and 1.0 μg / mL of casein peptide (SEQ ID NO.1).

[0119] IL-1β test results are as follows Figure 10 As shown, the IL-1β production in the model group suggests that lipopolysaccharide induces BV2 cells to release more IL-1β, while the IL-1β concentration decreased in all intervention groups. In the three dosage groups of casein peptide (SEQ ID NO.1), IL-1β concentration decreased with increasing peptide concentration. All of the above evidence indicates that the peptide can improve neuroinflammation.

[0120] In summary, the experimental results demonstrate that casein peptide (SEQ ID NO.1) possesses both anti-oxidative damage and anti-neuroinflammatory capabilities in BV2 cells. 1.0 μg / mL of casein peptide (SEQ ID NO.1) showed comparable efficacy to 5 μM curcumin in improving ROS, MDA, IL-1β, and NO levels. As is well known to those skilled in the art, BV2 cells are a commonly used in vitro model in neuroscience research, frequently used to study pathological processes such as neuroaging. BV2 cells are a microglia cell line derived from mice. The excellent antioxidant and anti-inflammatory capabilities of casein peptide (SEQ ID NO.1) in BV2 cells reflect its potential to improve cognitive function and delay neuroaging, suggesting broad application prospects in the treatment of neuroaging.

[0121] Example 8: Effect of casein peptides on relative telomere length of neural stem cells derived from SD rats

[0122] Neural stem cells derived from SD rats were used at a rate of 2×10⁻⁶. 5Casein peptide (SEQ ID NO.1) was cultured at a density of 0.01 μg / mL in T25 culture flasks, 5 mL per flask. Casein peptide (SEQ ID NO.1) was injected at final concentrations of 0.01 μg / mL, 0.1 μg / mL, and 1 μg / mL, with 5 μM curcumin as a positive control, for 10 consecutive days. After modeling with 20 μM tert-butanol hydroperoxide for 48 h, genomic DNA was extracted and uniformly diluted to 50 ng / μL. Relative telomere length was detected by qt-PCR, using the same methods, reagents, and primer sequences as in Example 5. The experiment was repeated three times, with each sample tested three times.

[0123] The results are as follows Figure 11 As shown, compared with the control group, neural stem cells in the model group shortened rapidly after treatment with tert-butanol hydrogen peroxide. In the three intervention groups pretreated with active peptides for 10 days, the relative telomere length was longer than that in the model group. The relative telomere lengths after intervention with 0.1 μg / mL and 1.0 μg / mL casein peptide (SEQ ID NO. 1) were statistically significantly different from those in the model group. The relative telomere length in the positive control group was also longer than that in the model group. These results indicate that casein peptide (SEQ ID NO. 1) can delay telomere wear.

[0124] Neural stem cells, derived from primary cells in the hippocampus of newborn rats, are the only cells in the nervous system capable of proliferation. Neural stem cells induced by tert-butanol peroxide are a commonly used in vitro model in neuroscience research, suitable for assessing telomere wear. The ability of casein peptide (SEQ ID NO.1) to delay telomere wear in neural stem cells reflects its potential to improve cognitive function and delay neuroaging, showing broad application prospects in the treatment of neuroaging.

[0125] This invention utilizes *Bifidobacterium longum* BNCC 185354 to ferment and prepare casein hydrolysate, which possesses the ability to improve and delay neurosensory aging. Further investigation revealed that the casein peptide (amino acid sequence shown in SEQ ID NO. 1) in this casein hydrolysate exhibits the highest bioactivity, possessing anti-neuroinflammatory, anti-neural cell oxidative damage, and telomere attenuation capabilities. This casein hydrolysate and the active peptide can be used to prepare drugs for the prevention and / or treatment of neurosensory aging, showing broad application prospects.

[0126] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A casein hydrolysate, characterized in that: The casein hydrolysate was obtained by hydrolyzing casein with Bifidobacterium longum BNCC 185354.

2. The casein hydrolysate according to claim 1, characterized in that, The casein hydrolysate contains one or more casein peptides with the following amino acid sequences: (1) SEQ ID NO.1; (2) SEQ ID NO.3; (3) SEQ ID NO.6; (4) SEQ ID NO.7; (5) SEQ ID NO.

8.

3. The method for preparing the casein hydrolysate according to claim 1, characterized in that, Includes the following steps: Step S1: Prepare a casein solution, wherein the casein solution is obtained by mixing casein and an inorganic base in water; Step S2: Mix the activated Bifidobacterium longum BNCC 185354 with the casein solution, ferment, centrifuge and collect the supernatant to obtain the casein hydrolysate.

4. The preparation method according to claim 3, characterized in that: In step S1, the mass ratio of casein to water is 2-3:97-98, and the mass ratio of inorganic alkali to water is 4-5:95-100.

5. The preparation method according to claim 3, characterized in that: In step S2, the amount of Bifidobacterium longum BNCC185354 added is (3-7) × 10⁻⁶. 8 CFU / mL.

6. A casein peptide, characterized in that: The amino acid sequence of the casein peptide is shown in SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO.

8.

7. The casein peptide according to claim 6, characterized in that: The amino terminus and / or carboxyl terminus of the casein peptide are modified.

8. The gene sequence encoding the casein peptide of claim 6.

9. The use of the casein hydrolysate of claim 1 or the casein peptide of claim 6 in the preparation of a medicament for preventing and / or treating neurosenescence, wherein the medicament possesses at least one of the following functions: (1) Clear reactive oxygen free radicals in nerve cells; (2) Extend the telomere length in the hippocampus; (3) Alleviate oxidative damage to DNA in the hippocampus; (4) Reduce the concentration of malondialdehyde in nerve cells; (5) Reduce the concentration of nitric oxide in nerve cells; (6) Reduce the concentration of interleukin-1β in nerve cells.

10. A drug for preventing and / or treating neurosenescence, characterized in that, The drug comprises the casein hydrolysate of claim 1 or the casein peptide of claim 6, and the drug has at least one of the following functions: (1) Clear reactive oxygen free radicals in nerve cells; (2) Extend the telomere length in the hippocampus; (3) Alleviate oxidative damage to DNA in the hippocampus; (4) Reduce the concentration of malondialdehyde in nerve cells; (5) Reduce the concentration of nitric oxide in nerve cells; (6) Reduce the concentration of interleukin-1β in nerve cells.

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