Anti-stress polypeptide composition and preparation method thereof

Abstract

The invention belongs to the technical field of polypeptide extraction, and relates to an anti-stress polypeptide composition and a preparation method thereof. A novel functional peptide group with a specific amino acid sequence combination (such as short peptides rich in tyrosine, proline and glutamine) is enriched from complex casein hydrolysate through an innovative separation strategy, the biological activity of the novel functional peptide group is remarkably superior to that of a traditional crude hydrolysate, and the novel functional peptide group belongs to a brand-new functional active substance for feed or veterinary medicine. Therefore, the traditional Chinese medicine composition not only can be used as an auxiliary treatment preparation in veterinary clinic for intervening stress, inflammatory bowel disease or immunosuppression and other diseases, but also can be used as a functional feed additive to be widely applied to daily ration of livestock and poultry and pets, the disease resistance and production performance of animals are improved, the market application prospect is wide, and the traditional Chinese medicine composition has important economic and social values.

Description

Technical Field

[0001] This invention belongs to the field of polypeptide extraction technology and relates to an anti-stress polypeptide combination and its preparation method. Background Technology

[0002] In the breeding, transportation, and daily management of livestock, poultry, aquatic animals, and laboratory animals, animals often experience significant stress responses due to external stimuli such as sudden environmental changes, excessive density, temperature fluctuations, noise interference, or human manipulation. This stress state activates the hypothalamic-pituitary-adrenal (HPA) axis, leading to the excessive secretion of stress hormones such as cortisol. This, in turn, causes immunosuppression, decreased feed intake, slowed growth rate, deterioration of meat quality, and a significant increase in susceptibility to pathogenic microorganisms, seriously affecting animal health and production efficiency.

[0003] Currently, commonly used anti-stress interventions in clinical practice mainly include adding sedative drugs, vitamin preparations, or glucocorticoid regulators and other chemically synthesized substances. However, these methods generally have problems such as drug residue risks, potential toxic side effects, disruption of intestinal microecological balance, or tolerance after long-term use, making it difficult to meet the requirements of modern animal husbandry for green, safe, and sustainable development.

[0004] In contrast, naturally derived functional bioactive peptides are gradually becoming a research hotspot for replacing traditional anti-stress products due to their high efficiency, low toxicity, lack of residue, and easy absorption. Among them, hydrolyzed casein, as an important derivative of milk protein, is rich in various bioactive fragments with neuromodulatory, immune-enhancing, and antioxidant activities, such as casein phosphopeptides (CPPs), γ-aminobutyric acid (GABA)-related peptides, and opioid-like bioactive peptides, which theoretically have the potential to relieve central nervous system tension, stabilize mood, and regulate autonomic nervous function.

[0005] However, although casein hydrolysates exhibit certain physiological functions, their complex composition and high peptide diversity mean that systematic screening, functional verification, and mechanism of action studies for specific animal stress models remain scarce. In particular, there is a lack of technical pathways for accurately identifying and enriching core functional peptides with true anti-stress activation from a vast peptide library, resulting in unstable efficacy, poor targeting, and unclear application effects of existing products. Therefore, there is an urgent need to establish an efficient, controllable, and scalable functional peptide preparation process, combined with molecular recognition and bioactivity evaluation methods, to directionally isolate key short peptides from hydrolyzed casein that can mimic neurotransmitter effects and regulate stress pathways, and to verify their actual anti-stress effects in animals. This would provide a theoretical basis and technical support for developing a new generation of safe and efficient natural anti-stress additives. Summary of the Invention

[0006] To address the shortcomings of the prior art, this invention aims to provide a method for preparing an anti-stress polypeptide combination. The polypeptides described in this invention are derived from food-grade casein and are obtained through enzymatic hydrolysis, separation, and purification, thus avoiding toxic byproducts that may be introduced during chemical synthesis. This method has good biosafety and is suitable for long-term clinical use in animals.

[0007] One objective of this invention can be achieved through the following technical solutions:

[0008] A method for preparing an anti-stress polypeptide combination, the method comprising the following steps:

[0009] S1. Dissolve casein in water and sonicate to obtain a protein solution, then add a complex enzyme for hydrolysis;

[0010] S2. Then, membrane separation is performed to obtain the intercepted liquid, and the intercepted liquid is then eluted and fractionated using gradient osmotic pressure to obtain small peptides.

[0011] S3. Selectively enrich the target anti-stress peptide in small peptides using targeted adsorption materials to obtain an desorption solution, then perform microbial modification to obtain a peptide solution, and finally stabilize and dry it.

[0012] In the above-mentioned method for preparing an anti-stress polypeptide combination, in step S1, casein is dissolved in water and the pH is adjusted to 6.1–6.3 using phosphate buffer.

[0013] Preferably, the ultrasonic treatment frequency is 20–25 kHz, the power density is 10–15 W / L, and the intermittent mode is used (working for 1 minute, pausing for 1 minute) for 10–15 minutes.

[0014] Further preferred options include a protein solution with ≥95% soluble protein, <20 NTU turbidity, pH 6.1–6.3, 7–10 wt% solids content, and a temperature controlled at 38–46 °C.

[0015] In the above-mentioned method for preparing an anti-stress polypeptide combination, the complex enzyme includes an alkaline protease and a flavor protease, wherein the amount of metal-dependent alkaline protease (Metalloprotease) added is 0.8–1.2% of the protein solution mass, and the amount of fungal flavor protease (Aspergillus oryzae) added is 0.3–0.7% of the protein solution mass.

[0016] In the above-mentioned method for preparing an anti-stress peptide combination, step S2, gradient osmotic elution and fractionation, specifically includes: establishing a four-stage osmotic pressure using glycerol or sorbitol, with the concentration gradients set sequentially as 0%, 5-15%, 18-23%, and 25-35% (w / w), the contact time for each stage being 15-30 min, and the temperature being 20-25℃.

[0017] This invention employs a four-stage gradient osmotic pressure elution design. By progressively increasing the concentration of glycerol or sorbitol, it enables the differentiated release and fractional purification of anti-stress peptides under mild conditions. This method not only improves the resolution and bioactivity retention of hydrolysates but also effectively reduces the risk of protein aggregation and loss of activity. Compared with traditional single-stage or high-salt elution, this process offers advantages such as being mild, highly selective, non-toxic, and industrially scalable.

[0018] In the above-mentioned method for preparing an anti-stress peptide combination, the targeted adsorption material is composed of functionalized magnetic nanoparticles coupled with GABA pseudoligands.

[0019] The targeted adsorption material used in this invention is composed of functionalized magnetic nanoparticles coupled with GABA ligands. This material possesses both biomimetic recognition and magnetic response properties, enabling it to specifically adsorb and enrich anti-stress peptides containing GABA receptor recognition sequences.

[0020] During adsorption, the GABA ligand reversibly binds to the target peptide through hydrogen bonds, electrostatic interactions, and hydrophobic interactions, ensuring a gentle separation process and preservation of activity. After adsorption, rapid solid-liquid separation can be achieved by applying an external magnetic field, avoiding activity loss caused by centrifugation and filtration. Mild elution of the target peptide and carrier regeneration can be achieved by adjusting the solution pH or osmotic pressure gradient.

[0021] Compared with traditional chromatographic separation, this targeted adsorption material has the advantages of high selectivity, fast separation speed, high reusability, and minimal damage to peptide structure. It can significantly improve the purity and specific activity of anti-stress peptides, and is an efficient, safe and industrially scalable separation and purification method.

[0022] In the above-mentioned method for preparing an anti-stress peptide combination, the method for preparing the targeted adsorption material includes the following steps: functionalized magnetic nanoparticles are modified by reflux with APTES ethanol solution, then crosslinked and activated with glutaraldehyde, and finally coupled with GABA pseudoligands.

[0023] The targeted adsorption material of this invention possesses uniform magnetic response characteristics and a high-density amine-modified layer, enabling targeted adsorption of anti-stress peptide molecules through the specific recognition of GABA ligands. Compared with traditional physical adsorption or non-specific resins, this material exhibits approximately 2–3 times higher selective enrichment efficiency, reduces non-specific adsorption by more than 40%, and demonstrates excellent reusability (adsorption capacity retention ≥90% after 5 adsorption-elution cycles).

[0024] In the above-mentioned method for preparing an anti-stress peptide combination, the functionalized magnetic nanoparticles are Fe3O4@SiO2.

[0025] In the above-mentioned method for preparing an anti-stress polypeptide combination, microbial modification is performed using exopeptidase preparations derived from Lactobacillus casei or lactic acid bacteria.

[0026] In the above-mentioned method for preparing an anti-stress peptide combination, the stabilization drying includes the following steps: adjusting the solid content of the peptide solution to 2-5% (w / v), then adding 0.3-0.8% (w / v) taurine and 0.02-0.05% antioxidant, stirring evenly, and then freeze-drying.

[0027] The present invention also provides a novel anti-stress polypeptide combination, which is prepared by the above method, wherein the polypeptide sequences are shown in SEQ.ID NO 1-5.

[0028] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0029] 1. The functional polypeptides described in this invention are derived from food-grade casein and are prepared through targeted enzymatic hydrolysis and efficient separation and purification technology. The entire process does not require chemical synthesis steps, avoiding the introduction of organic solvent residues and toxic byproducts, ensuring the high purity and excellent biocompatibility of the product, meeting food safety and veterinary drug safety standards, and suitable for long-term clinical application in livestock, pets and other animals, with significant advantages in biosafety.

[0030] 2. This invention employs synergistic hydrolysis of alkaline protease and flavor protease, combined with two-stage dynamic membrane separation (10kDa and 1–3kDa ultrafiltration), gradient osmotic pressure elution, and targeted enrichment of GABA receptor-mimicking ligand magnetic nanoparticles. The process is simple and the reaction conditions are mild (e.g., temperature controlled at 37–45℃, pH near neutral). This not only effectively protects the structural integrity of the active peptides but also provides good controllability, repeatability, and stability. The equipment used is all common industrial equipment, making it easy to achieve large-scale production, significantly reducing unit costs, and possessing good industrialization prospects.

[0031] 3. The peptides of this invention exhibit significant multiple biological activities, including antioxidant capacity by scavenging free radicals and enhancing the activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px); anti-inflammatory effects by inhibiting the NF-κB pathway and reducing the expression of pro-inflammatory factors such as TNF-α and IL-6; intestinal barrier repair function by promoting the secretion of intestinal mucin and the expression of tight junction proteins (such as ZO-1 and occludin); and immunomodulatory effects by activating macrophage phagocytic capacity and increasing IgA levels. In clinical application trials in dogs, cats, and weaned piglets, the addition of the peptides of this invention significantly alleviated diarrhea symptoms, improved fecal scores, increased daily weight gain, and improved feed conversion ratio, confirming its definite efficacy in preventing and treating digestive tract diseases and improving animal health.

[0032] 4. This invention utilizes an innovative separation strategy to enrich novel functional peptide groups with specific amino acid sequence combinations (such as short peptides rich in tyrosine, proline, and glutamine) from complex casein hydrolysates. These peptides exhibit significantly superior bioactivity compared to traditional crude hydrolysates, representing a novel type of functional feed or veterinary active substance. Therefore, this invention can not only serve as an adjunct therapeutic agent in veterinary clinical practice for the intervention of stress, inflammatory bowel disease, or immunodeficiency, but also be widely applied as a functional feed additive in livestock and pet diets to enhance animal disease resistance and production performance. It has broad market application prospects and significant economic and social value. Detailed Implementation

[0033] The following are specific embodiments of the present invention, which further describe the technical solution of the present invention, but the present invention is not limited to these embodiments.

[0034] The terms “comprising,” “including,” “having,” “containing,” or any other variations thereof, as used herein, are intended to cover a non-exclusive inclusion. For example, a composition, step, method, or article that includes the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such composition, step, method, or article.

[0035] The terms "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., used in this invention refer to a specific feature, structure, material, or characteristic described in connection with that embodiment or example, which is included in at least one embodiment or example of the invention. In this specification, the illustrative expressions of the above terms are not necessarily directed at the same embodiment or example. Furthermore, the technical features involved in the various embodiments of the invention can be combined with each other as long as they do not conflict with each other.

[0036] Among them, raw materials and auxiliary materials:

[0037] 1. Casein (food / pharmacopoeia grade, ≥90% protein, moisture ≤6%, ash ≤2%).

[0038] 2. Deionized water (conductivity ≤ 1 μS / cm).

[0039] 3. Enzyme preparations (food / pharmaceutical grade):

[0040] Alkaline protease (activity ≥ 2.0 × 10⁻⁶) 5 (U / g) purchased from the company's Bacillus thermoproteolyticus alkaline protease Thermolysin;

[0041] Flavor protease (≥5.0×10) 4U / g) purchased from Aspergillus oryzae (Novozymes) Protease.

[0042] 4. Probiotics Lactobacillus casei or L. plantarum (≥10) 9 CFU / g (food grade).

[0043] 5. Targeted adsorption materials: Magnetic nanoparticles Fe3O4@SiO2 (average particle size 80–150 nm, saturation magnetization ≥50 emu / g); Surface activation and coupling reagents: APTES (3-aminopropyltriethoxysilane), glutaraldehyde or EDC / NHS; GABA receptor mimicry ligands: peptide / amine "ligand-like" γ-aminobutyric acid derivatives (Ala-Gly-GABA terminal -NH2 facilitates coupling);

[0044] The preparation of targeted adsorption materials includes the following steps:

[0045] S1. Functionalized magnetic nanoparticle modification: 1.00 g of functionalized magnetic nanoparticles (Fe3O4@SiO2 matrix) were dispersed in 50 mL of anhydrous ethanol, and 10 mL of 2% (v / v) APTES ethanol solution was added. The mixture was reacted at reflux temperature (approximately 80 °C) for 3 h. After the reaction was completed, the particles were collected by magnetic separation and washed three times successively with ethanol and deionized water, and then dried under vacuum to obtain silanized modified magnetic nanoparticles.

[0046] The preferred concentration of the APTES ethanol solution is 2% (v / v), which can be adjusted within the range of 0.5-5% (v / v) to regulate the surface amine group density.

[0047] S2. Glutaraldehyde Activation Treatment: The above-mentioned silanized modified magnetic nanoparticles were dispersed in 50 mL of 2.5% (v / v) glutaraldehyde aqueous solution and reacted with magnetic stirring at room temperature (20–25℃) for 1 h. After the reaction, the supernatant was thoroughly washed with deionized water until there was no free glutaraldehyde odor and was ready for use.

[0048] S3, GABA ligand coupling: Magnetic nanoparticles activated with glutaraldehyde were added to 20 mL of PBS buffer (pH 7.4) containing 0.03 g of GABA ligand. The mixture was stirred gently at room temperature for 8 h. After the reaction was completed, the solid was collected by magnetic separation and washed twice with PBS and once with deionized water. The target adsorption material was obtained by vacuum drying.

[0049] 6. Osmotic Elution System

[0050] Glycerol or sorbitol (food grade, ≥99%); NaCl; PBS buffer (pH 7.4).

[0051] 7. Stabilization-associated substances

[0052] Taurine (≥99%) or β-alanine (≥99%); supplemented with the antioxidant vitamin C.

[0053] 8. Buffer solutions and conventional chemicals

[0054] Phosphate buffer (0.02–0.05M, pH 6.2 / 7.4 formulations); pH adjustment with NaOH / HCl; ethanol.

[0055] Main equipment:

[0056] 1. Jacketed enzymatic hydrolysis reactor (corrosion resistant, with online pH / temperature probe, 5-50L experimental; 100-500L pilot-scale).

[0057] 2. Dynamic cross-flow membrane system: Two-stage membrane modules (polyethersulfone or regenerated cellulose) with a 10kDa and 1-3kDa transmembrane pressure (TMP) of 0.5-1.5 bar.

[0058] 3. Permanent magnet separator / magnetic frame (can be scaled up to a pipeline magnetic separator).

[0059] 4. Pulsed electric field (PEF) device (optional enhancement: field strength 15-30kV / cm, pulse width 20-50μs, frequency 1-5Hz).

[0060] 5. High-speed refrigerated centrifuge; constant temperature oscillator; ultrasonic processor (20-40kHz).

[0061] 6. Aseptic fermenter / shaker (for co-culturing probiotics).

[0062] 7. Vacuum freeze dryer (cold trap -50~-80℃, chamber ≤50Pa).

[0063] 8. HPLC / UPLC (C18 column, UV 214 / 220nm); LC–MS (optional); Total nitrogen / amino nitrogen analyzer; ACE inhibitory activity assay device.

[0064] Example 1:

[0065] S1. First, add casein to deionized water at a concentration of 8% (w / v) and stir at 42°C for 40 minutes to ensure thorough dispersion and swelling. Adjust the pH of the system precisely to 6.2 using 0.03M phosphate buffer to maintain the optimal acid-base environment for subsequent enzymatic hydrolysis. To promote casein dissolution, ultrasonic-assisted treatment can be used, with parameters set at a frequency of 23kHz and a power density of 12W / L, in intermittent mode (1 minute on, 1 minute off), for a total treatment time of 12 minutes. Throughout the pretreatment stage, ensure the substrate is homogeneous and free of lumps, ultimately achieving a quality standard of ≥95% soluble protein and <20 NTU turbidity. Key process parameters (CPP) include: pH controlled at 6.2, solids content maintained at approximately 8%, and temperature controlled at 42°C to ensure efficient initiation of the subsequent enzymatic reaction.

[0066] S2. A complex enzyme system was added to the pretreated casein solution, with alkaline protease added at 1.0% of the protein solution mass, initially dominating the hydrolysis reaction. After 2 hours of reaction, 0.5% of the protein solution mass of flavor protease was added to optimize peptide flavor and reduce bitterness. The reaction was carried out at 40℃ for 5 hours, with the pH strictly controlled at 6.2, dynamically adjusted by online automatic addition of NaOH or HCl. The stirring speed was set to 300 rpm, with a recommended DoE optimization value of 300 rpm to ensure uniform mass transfer and avoid shear damage. Pulsed electric field (PEF) enhancement technology was introduced: field strength 23 kV / cm, pulse width 30 μs, frequency 1 Hz.

[0067] The hydrolysis endpoint is determined based on HPLC detection results: the peak area of ​​1–3 kDa soluble oligopeptides accounts for ≥60% of the total peptide peak area, or the degree of protein hydrolysis (DH) reaches 10–15%. After reaching the endpoint, immediately raise the temperature to 82°C and hold for 10 minutes to inactivate the enzyme and prevent excessive hydrolysis and Maillard browning.

[0068] The key process parameters (CPP) are a reaction temperature of 40℃, pH 6.2, and DH controlled at 10–15%; the quality standard (CQA) requires that the bitterness threshold index not exceed the standard and the content of volatile aldehyde / ketone byproducts be low.

[0069] S3. After enzyme inactivation and cooling, the hydrolysate enters a two-stage dynamic membrane separation system. The first stage uses a 10kDa ultrafiltration membrane for external filtration, with the operating temperature controlled at 20–25℃, transmembrane pressure differential (TMP) set at 1.0 bar, and cross-flow velocity at 1.2 m / s. This primarily removes unhydrolyzed large molecular weight proteins and other proteins, and the permeate is collected for the next stage of treatment. The second stage uses a 2kDa membrane to enrich the permeate from the first stage, similarly controlling the TMP at 0.8 bar and the cross-flow velocity at 1.2 m / s. The retentate is collected, achieving effective enrichment of target short peptides (especially in the 300–1500 Da range). After each batch operation, CIP washing is performed for 30 minutes at 45℃ using 0.1M NaOH solution, followed by rinsing with pure water until the effluent pH is close to 7.

[0070] The Quality Assurance (CQA) standard requires that the enrichment of the target 1-3kDa peptide peaks be increased by ≥2.0 times, and the area of ​​residual macromolecular impurities be <5%.

[0071] S4. The retentate is sequentially diluted at a 1:4 volume ratio into 0%, 10%, 20%, and 30% (w / w) glycerol gradient solutions. Osmotic pressure changes disrupt the weak interactions between peptides and polysaccharides or micro-groups, promoting the release of bound small peptides. Each stage is gently stirred at 23°C for 20 minutes (contact time ≥15 minutes), and the supernatant is collected after settling. The supernatants from each stage are dialyzed through a 1 kDa dialysis bag to remove glycerol, obtaining the small peptide fraction. Strict control of the glycerol concentration deviation (≤±1%) is necessary during the operation to avoid affecting the fractionation results.

[0072] The Quality Standard (CQA) focuses on the F2 and F3 fractions, requiring optimal enrichment of small peptide peaks in the 300-1500 Da range, and a conductivity recovery to <2 mS / cm after deglycerolization, indicating effective removal of small molecule impurities.

[0073] S5. Specific enrichment was achieved using functionalized magnetic nanoparticles (Fe3O4@SiO2). First, the magnetic beads were refluxed with 2% APTES ethanol solution for 2 hours to introduce amino groups, followed by cross-linking with 2.5% glutaraldehyde for 1 hour to activate the aldehyde groups. Then, a GABA-like ligand with a terminal -NH2 group was dissolved in PBS buffer (pH 7.4) and incubated with the activated magnetic beads at room temperature for 2 hours to complete ligand coupling. Unreacted aldehyde groups were then blocked with 0.5M ethanolamine. Using small peptide fractions as raw materials, the mixture was added at a magnetic bead to solution volume ratio of 1:200 and gently incubated at pH 7.3, 80mM NaCl, and 25°C for 40 minutes to selectively bind the target anti-stress peptide. After binding, the mixture was washed twice with PBS to remove non-specific adsorbed substances.

[0074] The pH was adjusted to 5.8 with acetate buffer, and the mixture was allowed to react for 8 minutes to achieve a gentle release. Finally, an external magnetic field was applied for 1.5 minutes to complete magnetic separation, and the eluent was collected.

[0075] The Quality Standard (CQA) requires a target peptide recovery rate of ≥70%, a purity improvement of ≥15 percentage points, and no Fe leaching from the magnetic beads (<5ppm).

[0076] S6. After sterilizing the eluent by filtration through a 0.22μm filter, inoculate with Lactobacillus casei to a final concentration of 10. 7 -10 8 CFU / mL, co-cultured at 30–37℃ and 80–120 rpm for 30–120 minutes; this process aims to selectively degrade non-target long-chain heteropeptides and increase the proportion of short peptides.

[0077] The Quality Assurance (CQA) requires a ≥50% reduction in the relative content of low-value long peptides and a significant increase in the peak area of ​​target short peptides. If used in food or pharmaceuticals, it is also necessary to ensure that microbial residues meet safety standards.

[0078] S7. Adjust the solid content of the modified target peptide solution to 3% (w / v), add 0.5% (w / v) of taurine as a protective agent and 0.03% (v) of vitamin C as an optional antioxidant, stir evenly, and then freeze dry.

[0079] The pre-freezing stage is maintained at -40℃ for 3 hours to ensure complete freezing; the sublimation drying stage controls the vacuum at 0.2 mbar, with the shelf temperature gradually increased from -20℃ to 0℃ over 10 hours; the desorption drying stage raises the temperature to 18℃ for 5 hours to completely remove bound water. The cold trap temperature must be maintained below -60℃, and the final vacuum must be stabilized at <0.3 mbar. The drying endpoint is determined by a moisture content ≤5% and a glass transition temperature Tg' > -20℃.

[0080] The resulting powder should be white, with a bulk density of 0.3 g / mL, and completely dissolve within 30 seconds. HPLC purity should be ≥90%. Key process parameters (CPP) include taurine addition, cold trap temperature, and vacuum level. Quality standards (CQA) cover appearance, solubility, purity, and stability indicators to ensure the product is suitable for use as a functional food or pharmaceutical ingredient.

[0081] Laboratory animals and grouping:

[0082] Sixty 6-week-old SPF-grade mice were randomly divided into 6 groups: a blank control group (distilled water), and one group each of P1, P2, P3, P4, and P5, with 10 mice in each group.

[0083] Administration method:

[0084] The medication was administered by gavage daily at a dose of 50 mg / kg for 28 consecutive days.

[0085] detection indicators

[0086] (1) Immune function: Detect serum IgG and IgA levels (ELISA method).

[0087] (2) Antioxidant capacity: Serum SOD, GSH-Px activity and MDA content were detected.

[0088] (3) Stress resistance: On day 20, mice were subjected to restraint stress test to observe changes in body weight and behavioral scores.

[0089] Experimental results:

[0090] P1 group.SEQ.ID NO 1 (LPYSG): Serum IgG increased by 36.4%, SOD activity increased by 28.7%, and MDA content decreased by 31.5%, showing the best immune enhancement and antioxidant effects.

[0091] P2 group.SEQ.ID NO 2(YGPVA): IgA increased by 22.6%, GSH-Px activity increased by 19.8%, moderate effect.

[0092] P3 group.SEQ.ID NO 3 (VPLSF): The main manifestation was improved stress resistance, and the weight loss was reduced by 40% compared with the control group.

[0093] P4 group.SEQ.ID NO 4 (GYPAL): The overall effect was average, with only a slight improvement in the antioxidant index.

[0094] P5 group.SEQ.ID NO 5(AVSLG): No significant improvement, close to the control level.

[0095] Conclusion: The overall results indicate that peptide P1 (LPYSG) exhibits the most significant immune-enhancing and antioxidant effects, outperforming other candidate peptides; P2 and P3 show some auxiliary effects, while P4 and P5 have weaker effects. Therefore, P1 (Lactopeptide-10) was ultimately selected as the optimal implementation scheme.

[0096] Example 2:

[0097] Functional testing:

[0098] ACE inhibition assay (HHL substrate): IC 50 Approximately 42 μM.

[0099] Antioxidant (DPPH): Scavenging rate 72% (0.5 mg / mL).

[0100] Stability: >80% retention rate in simulated gastric fluid after 60 min; >70% retention rate in simulated intestinal fluid after 120 min.

[0101] Example 3:

[0102] Blood pressure reduction and mechanism verification in spontaneously hypertensive rats (SHR)

[0103] Objective: To evaluate the antihypertensive effect of oral tyrosine-10 and its ACE-related mechanism in vivo.

[0104] Animals: Male SHR, 16–18 weeks old, n=40.

[0105] Grouping / Dosage (qd, 28 days, by gavage):

[0106] Vector control (0.5% CMC, n=10)

[0107] Low dose 3 mg / kg (n=10)

[0108] Medium dose 10 mg / kg (n = 10)

[0109] Positive control: captopril 10 mg / kg (n = 10)

[0110] Indicators and methods: Tail-wrap method + telemetry pressure measurement; daily 0 / 7 / 14 / 21 / 28; plasma Ang II, NO; aortic ACE activity; liver and kidney function; brain tissue antioxidant indicators (MDA, SOD).

[0111] Results (mean ± SEM):

[0112] Systolic blood pressure ΔSBP (relative to baseline, Day 28): Control -3.1±2.8 mmHg; 3 mg / kg -9.6±3.4 mmHg (p=0.018 vs Control); 10 mg / kg -17.8±3.9 mmHg (p<0.001); Captopril -21.5±4.1 mmHg (p<0.001).

[0113] Aortic ACE activity: Control 100%; 3 mg / kg 84.2% (p = 0.031); 10 mg / kg 68.7% (p < 0.001); captopril 61.3% (p < 0.001).

[0114] Plasma Ang II: Control 100%; 10 mg / kg 72.5% (p = 0.004). NO elevation: +22.1% (p = 0.012).

[0115] Oxidative stress: Brain MDA decreased by 18.4% (p = 0.026), and SOD activity increased by 21.7% (p = 0.019) (10 mg / kg).

[0116] Safety: There were no significant differences in weight, ALT / AST, CRE, and BUN compared with the control group; no behavioral abnormalities were observed.

[0117] Conclusion: Oral administration of tyrosine-10 produces dose-dependent hypotensive and ACE-inhibiting effects in SHR and improves oxidative stress.

[0118] Example 4: Anxiety / Sleep-Related Behaviors in Mice and Corticosterone

[0119] Animals: ICR mice (half male and half female), n=60.

[0120] Grouping / Dosage: Control, Tyrosine-10 (5 / 20 mg / kg, qd, 14 days), Positive Control (melatonin 10 mg / kg).

[0121] Assessment: Elevated cross maze (EPM), open field (OFT), sleep induction (sub-petrobarbital threshold); last blood sample for corticosterone measurement.

[0122] Results: EPM open arm dwell time (%): Control 21.4±3.2; 5mg / kg 27.9±3.6 (p=0.041); 20mg / kg 33.6±3.9 (p=0.003); Positive 35.1±4.1.

[0123] OFT central zone time increase: 20 mg / kg + 28% (p = 0.015).

[0124] Shortened sleep latency: 20 mg / kg - 19.6% (p = 0.009).

[0125] Serum corticosterone: 20 mg / kg - 23.4% (p = 0.012).

[0126] No abnormal motor coordination or body temperature was observed.

[0127] Conclusion: Tyrosine-10 exhibits anxiolytic and sleep-promoting signals in mice, accompanied by a decrease in corticosterone.

[0128] Example 5: Tolerance and pharmacokinetics of repeated 28-day administration in Beagle dogs

[0129] Animals: Beagle dogs, n=12 (half male and half female).

[0130] Dosage: 0 (carrier), 5, 25 mg / kg, qd, po, for 28 days, plus a 14-day recovery period for observation.

[0131] Endpoints: general condition, weight, food intake, ECG, blood pressure, hematology / biochemistry, urinalysis; sparse sampling pharmacokinetic (U-HPLC-MS) was performed on days 1 and 28.

[0132] Main results:

[0133] There were no deaths or drug-related clinical signs; no adverse changes were observed in ECG or blood pressure.

[0134] Mild fluctuations in blood biochemistry were within the physiological range; no dose-related toxicity was observed.

[0135] PK (day 28, 25 mg / kg): Cmax 1.8±0.4 μg / mL, Tmax 0.8 h, t1 / 2 2.3±0.6 h, AUC0-∞ 5.9±1.1 μg·h / mL; no significant accumulation was observed (Racc≈1.1).

[0136] NOAEL: ≥25mg / kg (oral, 28 days).

[0137] Conclusion: The oral administration was well tolerated in dogs, and the oral bioavailability and half-life were favorable for qd administration.

[0138] Example 6: Preclinical Toxicology Supplement

[0139] Acute toxicity (SD rats, po): No deaths occurred at the maximum tolerated dose (MTD) >2000 mg / kg; no gross or pathological abnormalities were observed in major organs.

[0140] Genetic toxicity: Ames test (5 strains with / without S9) negative; micronucleus test negative.

[0141] Safety pharmacology: hERG current inhibition rate <5% (50μM); no effect on mouse Rotarod.

[0142] Example 7: Companion Animal Clinical Trial I – Elderly Hypertensive Cats

[0143] Design: prospective, randomized, double-blind, placebo-controlled, multicenter; ethical approval and informed consent obtained from the participants.

[0144] Enrollment: Domestic cats n=36 (≥8 years old, persistent hypertension, Doppler SBP 160–200 mmHg; allowed for stable control of CKD stage 2–3).

[0145] Grouping / Dosage:

[0146] Placebo (n=12);

[0147] Tyrosine-10 2mg / kg qd (n=12);

[0148] Tyrosine-10 6mg / kg qd (n=12);

[0149] Treatment duration: 28 days; patients with a previous stable dose of amlodipine are allowed to enroll, but must be randomized and stratified (with equal proportions in each group), with the investigational drug as an adjunct.

[0150] Primary endpoint: Day 28 SBP change from baseline.

[0151] Secondary endpoints: response rate (SBP decrease ≥15 mmHg or <160 mmHg), microalbumin / creatinine ratio, owner questionnaire (short version of cat anxiety score), and safety (liver and kidney function).

[0152] Results (mITT, mean ± SD): ΔSBP (mmHg): placebo -6.2 ± 10.7; 2 mg / kg -12.9 ± 12.1 (p = 0.046 vs placebo); 6 mg / kg -18.4 ± 11.3 (p = 0.004).

[0153] Response rate: 25.0% (placebo) vs 41.7% (2 mg / kg, p = 0.21) vs 66.7% (6 mg / kg, p = 0.012).

[0154] UA / Cr: 18% decrease in the 6 mg / kg group (p = 0.038).

[0155] Anxiety-like behavior scores improved by 22% in the 6 mg / kg group (p = 0.031).

[0156] Safety: There were no statistically significant differences in ALT / CRE / BUN among the three groups; two cases of transient loose stools (possibly related) did not require discontinuation of medication.

[0157] Conclusion: In older hypertensive cats, tyrosine-10 as adjunctive therapy can significantly reduce SBP and is well tolerated.

[0158] Example 8: Companion Animal Clinical Trial II - Canine Situational Anxiety (Noise Fear)

[0159] Design: Randomized, double-blind, positive control (comparison with L-theanine 10 mg / kg), parallel group; n = 45. Population: Medium to large breed dogs with a history of high sensitivity to fireworks / thunderstorms (confirmed by owner and veterinarian), and whose baseline behavior was within the target range for two weeks.

[0160] Administration:

[0161] Vector control (n=15)

[0162] Tyrosine-10 5mg / kg (n=15)

[0163] L-Theanine 10 mg / kg (n = 15)

[0164] Assessment: induced audio exposure + family natural event; heart rate, wearable activity meter, salivary cortisol (60 min before and after exposure), owner rating (0–10).

[0165] Results: Owner scores improved (the greater the decrease, the better): Control -1.1±1.6; L-theanine -2.4±1.8 (p=0.021); Tyrosine-10 -3.0±1.7 (p=0.003 vs. control; difference from L-theanine p=0.19).

[0166] Decreased area under the curve (AUC) of cortisol: Control 100%; L-theanine 86% (p = 0.047); Tyrosine -1078% (p = 0.006).

[0167] Peak heart rate: Control 165±18 bpm; Tyrosine-10 149±16 (p=0.014).

[0168] Safety: Mild somnolence 2 / 15 (acceptable), no serious gastrointestinal adverse events observed.

[0169] Conclusion: Tyrosine-10 showed a trend of improving situational anxiety and reducing physiological stress indicators.

[0170] The embodiments described herein cover any points not exhaustively within the scope of the technical claims of this invention, as well as new technical solutions formed by equivalent substitutions of one or more technical features in the embodiments. These are all within the scope of the claims of this invention. Furthermore, in all listed or unlisted embodiments of this invention, each parameter in the same embodiment merely represents an instance (i.e., a feasible solution) of its technical solution, and there is no strict coordination or limitation relationship between the parameters. The parameters can be substituted for each other without violating axioms and the claims of this invention, unless otherwise stated.

[0171] The technical means disclosed in this invention are not limited to those described above, but also include technical solutions composed of any combination of the above technical features. The above descriptions are specific embodiments of this invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of this invention, and these improvements and modifications are also considered within the scope of protection of this invention.

[0172] The specific embodiments described herein are merely illustrative of the spirit of the invention. Those skilled in the art to which this invention pertains may make various modifications or additions to the described specific embodiments or use similar methods to substitute them, without departing from the spirit of the invention or exceeding the scope defined by the appended claims.

Claims

1. A method of preparing a combination of anti-stress polypeptides, characterized in that, The method comprises the following steps: S1, dissolving casein in water for ultrasonic treatment to obtain a protein solution, and then adding a complex enzyme for hydrolysis; S2, then performing membrane separation to obtain a retentate, and then using gradient osmotic pressure elution fractionation to obtain small peptides; S3, using a targeted adsorption material to selectively enrich target anti-stress peptides in the small peptides to obtain a desorption solution, then performing microbial modification to obtain a peptide solution, and finally performing stabilization and drying.

2. The method for preparing an anti-stress polypeptide combination according to claim 1, characterized in that, In step S1, the casein is dissolved in water, and the pH is adjusted to 6.1-6.3 by using a phosphate buffer.

3. The method for preparing an anti-stress polypeptide combination according to claim 1, characterized in that, The complex enzyme comprises alkaline protease and flavor protease, wherein the alkaline protease is added in an amount of 0.8-1.2% of the mass of the protein solution, and the flavor protease is added in an amount of 0.3-0.7% of the mass of the protein solution.

4. The method for preparing an anti-stress polypeptide combination according to claim 1, characterized in that, In step S2, the gradient osmotic pressure elution fractionation specifically comprises the following steps: using glycerol or sorbitol to establish a four-stage osmotic pressure, and setting the concentration gradient in the order of 0%, 5-15%, 18-23%, and 25-35% (w / w), and the contact time of each stage is 15-30 min, and the temperature is 20-25°C.

5. The method for preparing an anti-stress polypeptide combination according to claim 1, characterized in that, The targeted adsorption material is coupled from functionalized magnetic nanoparticles and a GABA mimic ligand.

6. The method for preparing an anti-stress polypeptide combination according to claim 1, characterized in that, The preparation method of the targeted adsorption material comprises the following steps: refluxing and modifying the functionalized magnetic nanoparticles in an APTES ethanol solution, cross-linking and activating the functionalized magnetic nanoparticles with glutaraldehyde, and finally coupling the functionalized magnetic nanoparticles with the GABA mimic ligand.

7. The method of claim 6, wherein the anti-stress polypeptide combination is prepared by, The functionalized magnetic nanoparticles are Fe3O4@SiO2.

8. The method for preparing an anti-stress polypeptide combination according to claim 1, characterized in that, The microbial modification is performed by using an exopeptidase preparation from Lactobacillus casei or lactic acid bacteria.

9. The method for preparing an anti-stress polypeptide combination according to claim 1, characterized in that, The stabilization and drying comprise the following steps: adjusting the solid content of the peptide solution to 2-5% (w / v), then adding 0.3-0.8% (w / v) taurine and 0.02-0.05% antioxidant, uniformly stirring, and then performing freeze-drying.

10. A novel anti-stress polypeptide combination, wherein the polypeptide combination is prepared by the method according to any one of claims 1-9, and the sequence of the polypeptide combination is shown in SEQ. ID NO 1-5.