A cubilose peptide composition for activating long-life gene SIRT1 and a targeted enzymatic preparation method and application thereof

Abstract

The present application relates to the technical field of bioactive peptide preparation, and discloses a bird's nest peptide composition for activating long-life gene SIRT1 and a directional enzymatic preparation method and application thereof.The bird's nest peptide composition comprises four peptide segments shown in SEQ ID NO:1 to SEQ ID NO:4 with a molecular weight of 100-500 Da.The method comprises the following steps: subjecting bird's nest raw materials to pretreatment, directional enzymatic hydrolysis of composite enzymes, membrane concentration and spray drying to obtain intermediate peptides with a molecular weight of less than 3 kDa, and then subjecting the intermediate peptides to separation and purification to finally obtain the bird's nest peptide composition with a target molecular weight range.The present application solves the problems of wide molecular weight distribution and insufficient functional targeting of traditional bird's nest peptides by controlling the molecular weight range and the enzymatic hydrolysis process.The bird's nest peptide composition can significantly up-regulate the expression of aging genes in a zebrafish aging model and improve oxidative stress indicators, and has excellent anti-aging activity.

Description

Technical Field

[0001] This invention relates to the field of bioactive peptide preparation technology, specifically to a bird's nest peptide composition that activates the longevity gene SIRT1, its directional enzymatic hydrolysis preparation method, and its application. Background Technology

[0002] Bird's nest, a traditional and precious tonic, derives its nutritional value primarily from active ingredients such as sialic acid, protein, and epidermal growth factor. However, the proteins in bird's nest are mostly large molecules, resulting in low bioavailability upon direct consumption and limiting their effective function. To improve absorption and unlock deeper bioactivity, current technologies often employ enzymatic hydrolysis to break down bird's nest proteins into smaller peptides. These smaller peptides typically exhibit superior antioxidant and anti-inflammatory properties compared to the original protein, making them a hot research topic in the deep processing of bird's nest.

[0003] Meanwhile, research into the molecular mechanisms of aging is becoming increasingly in-depth, with the core regulatory role of longevity gene families (such as SIRT1) gradually becoming clear. SIRT1, as a type of NAD+, + SIRT1-dependent deacetylases play a crucial role in delaying cellular and tissue aging by regulating pathways such as cellular metabolism, DNA repair, and oxidative stress response. Therefore, identifying natural product active ingredients that can efficiently and specifically activate SIRT1 is of great significance for developing anti-aging functional products.

[0004] However, current production and application of bird's nest peptides still face significant limitations: First, traditional enzymatic hydrolysis processes often employ single enzymes or random hydrolysis, resulting in a wide molecular weight distribution of the peptide products (typically 1-10 kDa). This leads to low levels and unstable activity of active peptides with specific targeting functions (such as SIRT1 activation). Second, evaluations of the efficacy of bird's nest peptides largely rely on indirect indicators such as in vitro antioxidant activity, lacking verification of their direct molecular mechanisms regulating key aging pathways (such as SIRT1). This results in unclear product function claims and difficulty in increasing added value. Furthermore, known natural SIRT1 activators (such as resveratrol) generally suffer from low bioavailability and short half-life.

[0005] Therefore, there is an urgent need in this field for a method that can target and prepare bird's nest active peptides with a clear anti-aging mechanism (especially activation of SIRT1) in order to break through the bottleneck of existing technologies and promote the upgrading of bird's nest products from traditional tonics to precise functional raw materials. Summary of the Invention

[0006] This application aims to at least partially address one of the technical problems existing in the prior art. To this end, this application provides a bird's nest peptide composition that activates the longevity gene SIRT1.

[0007] This application is based on the inventor's following discoveries:

[0008] When the inventors conducted in-depth research on the enzymatic hydrolysis process of bird's nest protein, they found that the bird's nest peptides obtained by traditional single or random enzymatic hydrolysis methods have complex components and a wide molecular weight distribution (usually 1-10 kDa). Among them, the proportion of active peptides that can specifically act on specific biological targets (such as the longevity gene SIRT1) is low and the activity is unstable, resulting in unclear product functions and limited efficiency.

[0009] Unexpectedly, through extensive experiments, the inventors discovered that by employing a specific complex enzyme system of alkaline protease and trypsin, and under optimized enzyme dosage, temperature, and time parameters, targeted enzymatic hydrolysis of bird's nest, coupled with separation and purification by reversed-phase high-performance liquid chromatography under specific conditions, it is possible to stably and efficiently prepare bird's nest peptide components with molecular weights highly concentrated in the range of 100-500 Da. Furthermore, using techniques such as mass spectrometry, the inventors identified multiple characteristic amino acid sequences from this component, including SPLL, RGVF, PSGSF, and QLLL.

[0010] More importantly, by establishing a zebrafish embryonic aging model for functional verification, the inventors discovered and confirmed for the first time that the bird's nest peptide composition prepared by this method, with a molecular weight of 100-500 Da and containing the aforementioned characteristic sequences, can significantly upregulate the expression level of the SIRT1 gene in the aging model, and simultaneously systematically improve key oxidative stress and aging indicators such as SA-β-galactosidase activity, MDA content, SOD activity, and ROS levels, exhibiting clear and excellent anti-aging activity. This discovery reveals that by controlling a specific molecular weight range and preparation process, bird's nest protein can be directionally converted into active peptides with highly efficient SIRT1 pathway activation function, thereby solving the technical bottlenecks of poor functional targeting and unclear mechanisms of traditional bird's nest peptides.

[0011] In a first aspect, this application proposes a bird's nest peptide composition for activating the longevity gene SIRT1. According to embodiments of this application, the bird's nest peptide composition comprises four peptide segments as shown in SEQ ID NO: 1 to SEQ ID NO: 4. The bird's nest peptide composition according to embodiments of this application effectively overcomes the technical difficulties of complex composition, unclear active ingredients, and poor targeting in traditional bird's nest enzymatic hydrolysates. These specific oligopeptide sequences, after screening and identification, constitute key active sites for activating the longevity gene, exhibiting excellent bioavailability and cell penetration. They can not only precisely target and significantly upregulate the expression level of the longevity gene SIRT1, but also effectively inhibit the transcription of aging marker genes p53 and p21, and synergistically enhance the body's SOD enzyme activity to scavenge free radicals, thereby achieving highly efficient anti-aging through a dual mechanism of gene regulation and anti-oxidative stress. Simultaneously, establishing these specific sequences as characteristic components provides a clear material basis and detection indicators for the quality standardization control and efficacy evaluation of bird's nest peptide products.

[0012] In some aspects of this application, the molecular weights of the four peptides shown in SEQ ID NO: 1 to SEQ ID NO: 4 are all between 100 and 500 Da.

[0013] In a second aspect of this application, a method for preparing the bird's nest peptide composition described in the first aspect of this application is provided. According to an embodiment of this application, the method includes: (1) pre-treating bird's nest raw materials to obtain a bird's nest homogenate; (2) enzymatically hydrolyzing the bird's nest homogenate to obtain a bird's nest hydrolysate; (3) membrane concentrating and drying the bird's nest hydrolysate to obtain an intermediate peptide with a molecular weight of less than 3 kDa; (4) separating and purifying the intermediate peptide, collecting components with a molecular weight of 100-500 Da, to obtain the bird's nest peptide composition. According to the method of this application embodiment, the method fully releases and homogenizes bird's nest protein through the optimized pretreatment in step (1), laying the foundation for subsequent efficient enzymatic hydrolysis; the specific complex enzyme-directed enzymatic hydrolysis in step (2) is the key, which can efficiently and specifically hydrolyze the large molecular protein of bird's nest into a mixture rich in target active peptides; the membrane concentration and drying in step (3) realizes the effective enrichment and stable storage of intermediate products; the reversed-phase high-performance liquid chromatography purification in step (4) can separate and purify with high precision according to molecular weight (100-500 Da), and finally obtain high-purity and high-activity target bird's nest peptide products. The entire process route is designed in a coordinated manner, realizing efficient, controllable and stable preparation of target active peptides with good reproducibility.

[0014] In some aspects of this application, in step (1), the pretreatment includes: pulverizing the bird's nest, soaking it in water for 1-3 hours, then simmering it in boiling water for 2-4 hours, and then homogenizing it using a colloid mill at a pressure of 15-45 MPa.

[0015] In some aspects of this application, in step (2), the enzymatic hydrolysis is performed at a temperature of 50-60°C for 1-4 h; and / or, the enzyme used in the enzymatic hydrolysis is a complex enzyme of alkaline protease and trypsin, and the total amount of the complex enzyme added is 3000-7000 U / g bird's nest protein.

[0016] In some aspects of this application, after enzymatic hydrolysis, sterilization is also performed at 100°C for 15-25 minutes.

[0017] In some aspects of this application, in step (3), the drying is carried out under conditions where the inlet air temperature is 160-180°C and the outlet air temperature is greater than 90°C; and / or, the drying is spray drying.

[0018] In some aspects of this application, in step (4), the separation and purification are achieved by at least one of reversed-phase high-performance liquid chromatography, ion exchange chromatography, gel filtration chromatography and ultrafiltration-chromatography.

[0019] In some aspects of this application, in step (4), the separation and purification is achieved by gradient elution using a C18 column with an aqueous solution containing 0.05%-0.15% trifluoroacetic acid as mobile phase A and an acetonitrile solution containing 0.05%-0.15% trifluoroacetic acid as mobile phase B.

[0020] In some aspects of this application, the gradient elution procedure is as follows: the proportion of mobile phase B is increased from 20% to 40% within 15 minutes, and then the proportion of mobile phase B is increased from 40% to 80% within 2 minutes.

[0021] Additional aspects and advantages of this application will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of this application. Attached Figure Description

[0022] The above and / or additional aspects and advantages of this application will become apparent and readily understood from the description of the embodiments taken in conjunction with the following drawings, in which:

[0023] Figure 1 The molecular weight distribution of the bird's nest peptide compositions prepared in Examples 1-3 of this application is shown in the gel permeation chromatography results.

[0024] Figure 2 The mass spectrum of the bird's nest peptide composition prepared in this application;

[0025] Figure 3 This is a graph showing the effect of different concentrations of H2O2 on the survival rate of zebrafish embryos in Example 5 of this application.

[0026] Figure 4 This is a bar chart showing the effect of the bird's nest peptide composition in Example 5 of this application on the activity of SA-β-galactosidase induced by H2O2 in zebrafish embryos;

[0027] Figure 5 This is a bar chart showing the effect of the bird's nest peptide composition in Example 5 of this application on the MDA content of H2O2-induced zebrafish embryos;

[0028] Figure 6 This is a bar chart showing the effect of the bird's nest peptide composition in Example 5 of this application on the SOD activity of H2O2-induced zebrafish embryos.

[0029] Figure 7 The bar chart and fluorescence diagram show the effect of the bird's nest peptide composition in Example 5 of this application on the ROS level of H2O2-induced zebrafish embryos.

[0030] Figure 8 This is a bar chart showing the effect of the bird's nest peptide composition in Example 5 of this application on the expression level of the SIRT1 gene in H2O2-induced zebrafish embryos;

[0031] Figure 9 This is a bar chart showing the effect of the bird's nest peptide composition in Example 5 of this application on the expression level of the p53 gene in H2O2-induced zebrafish embryos;

[0032] Figure 10 This is a bar chart showing the effect of the bird's nest peptide composition in Example 5 of this application on the expression level of the p21 gene in H2O2-induced zebrafish embryos. Detailed Implementation

[0033] The embodiments of this application are described in detail below. The embodiments described below are exemplary and are only used to explain this application, and should not be construed as limiting this application.

[0034] It should be noted that the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Therefore, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature. Furthermore, in the description of this application, unless otherwise stated, "multiple" means two or more.

[0035] In this document, the term "bird's nest peptide composition" refers to a mixture of low molecular weight peptides, or its purified components, prepared from swiftlet nest using the specific directional enzymatic hydrolysis and separation purification process described in this application, with a molecular weight primarily ranging from 100 to 500 Daltons (Da). Its core characteristics lie in a specific molecular weight range and / or the inclusion of characteristic amino acid sequences such as SPLL, RGVF, PSGSF, and QLLL.

[0036] In this document, the term "molecular weight 100-500 Da" refers to the molecular weight range of the peptides constituting the main component in the bird's nest peptide composition claimed in this application, which is between 100 and 500 Daltons. This range, determined by gel permeation chromatography, is one of the key parameters ensuring that the peptides are easily absorbed and possess specific biological activities.

[0037] In this document, the terms "amino acid sequence SPLL, RGVF, PSGSF, QLLL" refer to the primary structure of a specific short peptide identified from the bird's nest peptide composition described in this application by liquid chromatography-mass spectrometry. Each letter represents an amino acid (such as S-serine, P-proline, L-leucine, R-arginine, G-glycine, V-valine, F-phenylalanine, Q-glutamine).

[0038] In this article, the term "SIRT1 (Sirtuin 1) gene," also known as the longevity gene or silencing information regulator 1, is a gene encoding NAD1. + The gene is a histone deacetylase-dependent enzyme. Its expression product, SIRT1 protein, plays a central role in regulating cellular metabolism, stress response, genome stability, and the aging process. In this application, SIRT1 activation is a key molecular target and mechanistic indicator for evaluating the anti-aging activity of the bird's nest peptide composition.

[0039] In this document, the term "targeted enzymatic hydrolysis" refers to a process in which one or more proteases are intentionally selected to hydrolyze bird's nest protein under optimized temperature, pH, time, and enzyme concentration in order to obtain functional peptides with a specific molecular weight range or specific sequence. In this application, it specifically refers to a hydrolysis process using a complex enzyme system of alkaline protease and trypsin.

[0040] In this document, the term "alkaline protease" refers to a protease that exhibits high activity under alkaline conditions (optimal pH 8-11), capable of hydrolyzing protein peptide bonds, and widely used in the production of small molecule peptides. In this application, it works synergistically with trypsin to achieve deep and specific hydrolysis of bird's nest protein.

[0041] In this document, the term "trypsin" refers to a serine protease that specifically hydrolyzes the peptide bonds at the carboxyl terminus of arginine or lysine. In this application, its use in conjunction with an alkaline protease helps to produce peptides within the target molecular weight range.

[0042] In this document, the term "enzyme addition of 3000-7000 U / g" refers to the total number of protease activity units added per gram of bird's nest protein raw material. "U" stands for enzyme activity unit, which, in the context of this application, is generally defined as the amount of enzyme required to catalyze the production of 1 micromolar product per minute from a substrate under specific conditions (temperature, pH). This parameter is crucial for controlling the degree of enzymatic hydrolysis and the properties of the product.

[0043] In this document, the term "reversed-phase high-performance liquid chromatography (RP-HPLC)" refers to an analytical / preparative chromatographic technique that separates solutes based on the difference in partition coefficients between a stationary phase (nonpolar, such as C18 bonded silica gel) and a mobile phase (polar, such as a water-acetonitrile solution containing trifluoroacetic acid). In this application, it is used for the fine separation of intermediate peptides of 1-3 kDa to enrich target peptides of 100-500 Da.

[0044] In this article, the term "C18 column" refers to a commonly used reversed-phase chromatography stationary phase with octadecylsilane as the bonded phase. It is highly hydrophobic and suitable for the separation of moderately polar molecules such as peptides.

[0045] In this document, the term "gradient elution" refers to an elution method in which the composition of the mobile phase (e.g., the acetonitrile ratio) is programmed to change over time during chromatographic separation. This application employs a specific gradient program (e.g., 20% → 40% → 80% acetonitrile) to achieve effective separation of peptides of different polarities.

[0046] In this document, the term "spray drying" refers to a unit operation that atomizes liquid material into fine droplets, contacts them with hot air, and instantly evaporates moisture to obtain a dry powder. In this application, it is used to rapidly dry a concentrated enzymatic hydrolysate of bird's nest into an intermediate peptide powder of 1-3 kDa.

[0047] In this document, the term "gel permeation chromatography (GPC) / gel filtration chromatography" refers to a chromatographic technique that separates solute molecules based on differences in solute molecular size (hydrodynamic volume) for determining the molecular weight and distribution of polymers. This application uses the method specified in GB / T 22729-2008 to determine the molecular weight distribution of a bird's nest peptide composition.

[0048] In this document, the term "liquid chromatography-mass spectrometry (LC-MS / MS)" refers to an analytical technique that combines the separation capabilities of liquid chromatography with the structural identification capabilities of mass spectrometry. In this application, it is used to separate complex mixtures of bird's nest peptides and to identify their amino acid sequences through mass spectrometric analysis of peptide precursor ions and fragment ions.

[0049] In this paper, the term "zebrafish aging model" refers to an experimental system that uses zebrafish embryos or adult zebrafish as research subjects and induces aging-related phenotypic and molecular changes in vivo through exogenous oxidative stressors (such as hydrogen peroxide, H2O2). This application utilizes this model to evaluate the anti-aging efficacy of bird's nest peptides.

[0050] In this paper, the term "SA-β-galactosidase" refers to senescence-associated β-galactosidase, a classic biomarker of cellular senescence, whose activity is significantly increased in senescent cells. This application measures its activity to evaluate the effect of bird's nest peptides in delaying cellular senescence.

[0051] In this article, the term "MDA (malondialdehyde)" refers to one of the major end products of lipid peroxidation. Its content can reflect the severity of free radical attack on the body or cells and is a key indicator of oxidative stress damage.

[0052] In this article, the term "SOD (superoxide dismutase)" refers to an important endogenous antioxidant enzyme that catalyzes the dismutation of superoxide anion free radicals into hydrogen peroxide and oxygen, and is a key component of the body's antioxidant defense system. Its activity level reflects antioxidant capacity.

[0053] In this article, the term "ROS (Reactive Oxygen Species)" refers to a general term for a class of chemically reactive oxygen species produced by aerobic metabolism in living organisms, such as superoxide anions and hydroxyl radicals. Excessive ROS can lead to oxidative stress, damage biological macromolecules, and is closely related to aging.

[0054] In this paper, the term "qRT-PCR (quantitative real-time polymerase chain reaction)" refers to a molecular biology technique that uses real-time fluorescence signals to monitor and precisely quantify specific genes during DNA amplification. In this application, it is used to quantitatively analyze changes in the mRNA expression levels of aging-related genes such as SIRT1, p53, and p21.

[0055] In this article, the term "p53 gene" refers to an important tumor suppressor gene involved in cell cycle regulation, DNA repair, and apoptosis. Activation under stress can also induce cellular senescence.

[0056] In this article, the term "p21 gene" refers to one of the downstream target genes of p53, which encodes a cyclin-dependent kinase inhibitor that can cause cell cycle arrest and participate in the aging process.

[0057] This application discloses a bird's nest peptide composition that activates the longevity gene SIRT1, a method for preparing the bird's nest peptide composition, and related uses, which will be described in detail below.

[0058] Bird's nest peptide composition that activates the longevity gene SIRT1

[0059] In a first aspect, this application proposes a bird's nest peptide composition for activating the longevity gene SIRT1. According to embodiments of this application, the bird's nest peptide composition comprises four peptide segments as shown in SEQ ID NO: 1 to SEQ ID NO: 4. The bird's nest peptide composition according to embodiments of this application effectively overcomes the problems of complex composition, unclear active ingredients, and poor targeting of traditional bird's nest enzymatic hydrolysates. These specific oligopeptide sequences, after screening and identification, constitute key active sites for activating the longevity gene, exhibiting excellent bioavailability and cell penetration. They can not only precisely target and significantly upregulate the expression level of the longevity gene SIRT1, but also effectively inhibit the transcription of aging marker genes p53 and p21, and synergistically enhance the body's SOD enzyme activity to scavenge free radicals, thereby achieving highly efficient anti-aging through a dual mechanism of gene regulation and anti-oxidative stress. Simultaneously, establishing these specific sequences as characteristic components provides a clear material basis and detection indicators for the quality standardization control and efficacy evaluation of bird's nest peptide products.

[0060] SPLL (SEQ ID NO: 1);

[0061] RGVF (SEQ ID NO: 2);

[0062] PSGSF (SEQ ID NO: 3);

[0063] QLLL (SEQ ID NO: 4).

[0064] According to embodiments of this application, by defining specific amino acid sequences (SPLL, RGVF, PSGSF, QLLL), the core active substances of the bird's nest peptide composition described in this application are further clarified. These specific peptide sequences have been experimentally verified as key active ingredients for SIRT1 gene activation and anti-aging functions, ensuring that the product's active ingredients are clearly defined, its quality is controllable, and its efficacy is traceable.

[0065] According to embodiments of this application, the molecular weights of the four peptides shown in SEQ ID NO: 1 to SEQ ID NO: 4 are all between 100 and 500 Da. The bird's nest peptide composition according to embodiments of this application, with its specific and narrow molecular weight range (100-500 Da), ensures excellent small molecule absorption characteristics. Furthermore, peptides within this range have been found to have a high correlation and specificity with activating the longevity gene SIRT1, overcoming the shortcomings of traditional bird's nest peptides, which suffer from unclear active ingredients and poor functional targeting due to their wide molecular weight distribution (e.g., 1-10 kDa).

[0066] Method for preparing bird's nest peptide composition

[0067] In a second aspect of this application, a method for preparing the bird's nest peptide composition described in the first aspect of this application is provided. According to an embodiment of this application, the method includes: (1) pre-treating bird's nest raw materials to obtain a bird's nest homogenate; (2) enzymatically hydrolyzing the bird's nest homogenate to obtain a bird's nest hydrolysate; (3) membrane concentrating and drying the bird's nest hydrolysate to obtain an intermediate peptide with a molecular weight of less than 3 kDa; (4) separating and purifying the intermediate peptide, collecting components with a molecular weight of 100-500 Da, to obtain the bird's nest peptide composition.

[0068] According to the method of this application embodiment, the method fully releases and homogenizes bird's nest protein through the optimized pretreatment in step (1), laying the foundation for subsequent efficient enzymatic hydrolysis; the specific complex enzyme-directed enzymatic hydrolysis in step (2) is the key, which can efficiently and specifically hydrolyze the large molecular protein of bird's nest into a mixture rich in target active peptides; the membrane concentration and drying in step (3) realizes the effective enrichment and stable storage of intermediate products; the reversed-phase high-performance liquid chromatography purification in step (4) can separate and purify with high precision according to molecular weight (100-500 Da), and finally obtain high-purity and high-activity target bird's nest peptide products. The entire process route is designed in a coordinated manner, realizing efficient, controllable and stable preparation of target active peptides with good reproducibility.

[0069] According to an embodiment of this application, in step (1), the pretreatment includes: pulverizing the bird's nest, soaking it in water for 1-3 hours, then simmering it in boiling water for 2-4 hours, followed by homogenization using a colloid mill at a pressure of 15-45 MPa. According to an embodiment of this application, the optimized soaking and simmering conditions ensure sufficient dissolution and moderate denaturation of the bird's nest protein, which is beneficial for enzymatic hydrolysis. Homogenization using a colloid mill under specific high pressure can greatly reduce the particle size of the material, increase the contact area between the enzyme and the substrate, and significantly improve the efficiency and uniformity of subsequent enzymatic hydrolysis.

[0070] According to an embodiment of this application, in step (2), the enzymatic hydrolysis is performed at a temperature of 50-60℃ for 1-4 hours; and / or, the enzyme used in the enzymatic hydrolysis is a complex enzyme of alkaline protease and trypsin, and the total amount of the complex enzyme added is 3000-7000 U / g of bird's nest protein. According to an embodiment of this application, the combination of alkaline protease and trypsin plays a synergistic hydrolytic role, which can more effectively cleave bird's nest protein to generate a large number of peptides within the target molecular weight range. Precisely controlled enzyme addition, temperature, and time parameters are the core process guarantee for achieving targeted enzymatic hydrolysis, avoiding over-hydrolysis or under-hydrolysis, and thus stably obtaining a high proportion of 100-500 Da active peptides.

[0071] According to embodiments of this application, after enzymatic hydrolysis, the process further includes sterilization at 100°C for 15-25 minutes, for example, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, 25 minutes, or a range between these two values, such as 16-25 minutes or 17-25 minutes. According to embodiments of this application, the sterilization step can rapidly terminate the enzymatic reaction, prevent further changes in the product, and ensure the stability of the product's activity and composition.

[0072] According to embodiments of this application, the sterilized product is further subjected to cooling and centrifugation. According to embodiments of this application, cooling and centrifugation can effectively remove incompletely hydrolyzed impurities, enzyme denaturation precipitates, etc., preliminarily purifying the enzymatic hydrolysate and providing cleaner raw materials for subsequent concentration and purification steps.

[0073] According to an embodiment of this application, in step (3), the drying is achieved under the following conditions: the inlet air temperature is 160-180℃, for example, 160℃, 162℃, 164℃, 166℃, 168℃, 170℃, 172℃, 174℃, 176℃, 178℃, 180℃, or a range between the two, 162-180℃, 164-180℃, and the outlet air temperature is greater than 90℃; and / or, the drying is spray drying. According to an embodiment of this application, by using spray drying and operating under the aforementioned temperature conditions, rapid dehydration and solidification of intermediate peptides can be achieved, minimizing the degradation or inactivation of heat-sensitive active peptides during the drying process, which is beneficial for maintaining their biological activity and obtaining a powdered intermediate product with good flowability, easy storage, and easy subsequent processing.

[0074] According to an embodiment of this application, in step (4), the separation and purification is achieved by at least one of reversed-phase high-performance liquid chromatography, ion exchange chromatography, gel filtration chromatography and ultrafiltration-chromatography.

[0075] According to an embodiment of this application, in step (4), the separation and purification is achieved by using a C18 chromatographic column with an aqueous solution containing 0.05%-0.15% (e.g., 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, or a range between 0.06%-0.15% and 0.07%-0.15%) of trifluoroacetic acid as mobile phase A, and using an acetonitrile solution containing 0.05%-0.15% (e.g., 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.10%, 0.11%, 0.12%, 0.13%, 0.14%, 0.15%, or a range between 0.06%-0.15% and 0.07%-0.15%) of trifluoroacetic acid as mobile phase B, and performing gradient elution.

[0076] According to an embodiment of this application, the gradient elution procedure is as follows: the proportion of mobile phase B is increased from 20% to 40% within 15 minutes, and then increased from 40% to 80% within 2 minutes. According to an embodiment of this application, using a C18 column reversed-phase chromatography system, combined with a specific mobile phase containing trifluoroacetic acid and the optimized gradient elution procedure, efficient separation can be achieved based on subtle differences in peptide polarity. This condition is suitable for separating target active components with molecular weights of 100-500 Da from a mixture of 1-3 kDa intermediate peptides with high purity and high recovery, and is a key step in obtaining the final highly active product.

[0077] The following will explain the solution of this application with reference to embodiments. Those skilled in the art will understand that the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of this application. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0078] Example 1: Preparation of a bird's nest peptide composition activating SIRT1

[0079] 1. Materials and Instruments

[0080] Ingredients: Dried bird's nest from swiftlets (commercially available).

[0081] Enzyme preparations: alkaline protease (enzyme activity ≥200,000 U / g), trypsin (enzyme activity ≥250,000 U / g).

[0082] Main equipment: high-speed pulverizer, constant temperature water bath, colloid mill, high-speed centrifuge, ultrafiltration membrane concentration system, spray dryer, reversed-phase high-performance liquid chromatography system (equipped with C18 column and ultraviolet detector), freeze dryer.

[0083] 2. Preparation steps

[0084] (1) Pretreatment: Weigh 50 g of dried bird's nest raw material and pulverize it using a high-speed pulverizer. Place the pulverized bird's nest in 2.5 L of distilled water (material-to-liquid ratio 1:50 w / v) and soak it at room temperature for 2 hours to allow it to fully expand. Then, transfer the mixture to a boiling water bath and simmer for 3 hours. Homogenize the simmered gelatinous substance using a colloid mill at a working pressure of 30 MPa and adjust the pH of the slurry to 7.0 to obtain a uniform bird's nest homogenate.

[0085] (2) Targeted enzymatic hydrolysis: A complex enzyme consisting of alkaline protease and trypsin was added to the above bird's nest homogenate. The total amount of the complex enzyme added was 4000 U / g (based on the protein content in the bird's nest raw material). The mixture was placed in a constant temperature water bath at 50°C and continuously stirred for 2.5 hours for enzymatic hydrolysis.

[0086] (3) Enzyme inactivation and preliminary separation: After the enzymatic hydrolysis is completed, the mixture obtained in step (2) is quickly heated to 100°C and kept for 20 minutes to completely inactivate the protease. Then the solution is cooled to room temperature (about 25°C). At 4°C, it is centrifuged at 10,000 rpm for 20 minutes, and the supernatant is carefully collected to obtain the bird's nest enzymatic hydrolysate.

[0087] (4) Concentration and preliminary drying: The enzymatic hydrolysate of bird's nest obtained in step (3) was concentrated and desalted using an ultrafiltration membrane with a molecular weight cutoff of 3 kDa. The concentrate was then spray-dried with the following process parameters: inlet air temperature 170℃ and outlet air temperature >90℃. The dried powder, which is the intermediate bird's nest peptide with a molecular weight range mainly less than 3 kDa, was collected and placed in a desiccator for later use.

[0088] (5) Fine separation and purification: The intermediate peptide less than 3 kDa obtained in step (4) above was dissolved in ultrapure water, filtered through a 0.22 μm microporous membrane, and then separated and purified by reversed-phase high-performance liquid chromatography. Chromatographic conditions: Column: Zorbax SB-C18 (4.6 mm × 250 mm, 5 μm). Mobile phase: Phase A was an aqueous solution containing 0.1% (v / v) trifluoroacetic acid, and Phase B was an acetonitrile solution containing 0.1% (v / v) trifluoroacetic acid. Elution program: Gradient elution was used. Within 0-15 minutes, the proportion of Phase B increased linearly from 20% to 40%; within 15-17 minutes, the proportion of Phase B increased linearly from 40% to 80% and was maintained for 3 minutes. Flow rate: 1.0 mL / min; Detection wavelength: 214 nm; Column temperature: 30℃.

[0089] Based on the chromatogram, the fraction corresponding to the target peak was collected (preliminary experiments and mass spectrometry identification showed that this fraction mainly contained peptides with molecular weights of 100-500 Da). The collected fractions were combined, and after removing acetonitrile by rotary evaporation, they were freeze-dried to finally obtain a white powdery target bird's nest peptide composition, referred to as bird's nest peptide 1.

[0090] Example 2: Preparation of a bird's nest peptide composition activating SIRT1

[0091] Except for the parameter adjustment in the following steps, the other steps in this embodiment are the same as in Example 1: In step (2), the total amount of compound enzyme added is adjusted to 3000 U / g, the enzymatic hydrolysis temperature is maintained at 50℃, and the enzymatic hydrolysis time is extended to 4 hours to ensure sufficient enzymatic hydrolysis. Finally, the bird's nest peptide composition described in Example 2 is obtained, referred to as bird's nest peptide 2.

[0092] Example 3: Preparation of a bird's nest peptide composition activating SIRT1

[0093] Except for the parameter adjustments in the following steps, the remaining steps in this embodiment are the same as in Example 1: In step (2), the total amount of compound enzyme added is adjusted to 7000 U / g, the enzymatic hydrolysis temperature is adjusted to 60℃, and the enzymatic hydrolysis time is shortened to 1 hour. The bird's nest peptide composition described in Example 3 is finally obtained, referred to as bird's nest peptide 3.

[0094] Example 4: Molecular weight distribution and peptide identification of bird's nest peptide composition

[0095] 1. Determination of molecular weight distribution

[0096] The determination of molecular weight was performed according to the gel filtration chromatography method in the national standard GB / T 22729-2008, "Marine Oligopeptides". Chromatographic column: TSK Gel G2000 SWXI; mobile phase: 45% (v / v) acetonitrile aqueous solution (containing 0.1% trifluoroacetic acid); flow rate: 0.5 mL / min; detection wavelength: 214 nm; standards: cytochrome C (12384 Da), aprotinin (6511 Da), bacitracin (1450 Da), oxidized glutathione (651 Da), and Gly-Gly-Gly (189 Da). The standard curve equation for molecular weight versus retention time is: y = 0.0929962x 2 -1.92603x+12.0588, where y represents retention time in minutes and x represents molecular weight in Da. Based on the standard curve, the distribution ratio of peptides with different molecular weights in each hydrolysate was calculated using the area normalization method.

[0097] The results are as follows Figure 1 As shown, calculated using the area normalization method, the proportion of components with molecular weights in the range of 100-500 Da in the product of Example 1 was 91.20%. In the product of Example 2, the proportion of components with molecular weights in the range of 100-500 Da was 89.5%. In the product of Example 3, the proportion of components with molecular weights in the range of 100-500 Da was 90.8%. The data indicate that the process parameter ranges defined in this application (Examples 1-3) can effectively enrich the target small molecule peptides.

[0098] 2. Identification of characteristic peptide sequences

[0099] The sequence analysis of the target bird's nest peptide composition obtained in Example 1 was performed using liquid chromatography-tandem mass spectrometry.

[0100] 2.1 Sample pretreatment: The peptide solution was desalted by ultrafiltration and desalting column.

[0101] 2.2 Chromatographic conditions: A nano-liquid chromatography system with a C18 analytical column was used, employing a gradient separation with 0.1% formic acid aqueous solution and 0.1% formic acid-acetonitrile solution as the mobile phases. Specific conditions were as follows: Solution A was a 0.1% formic acid aqueous solution, and Solution B was a 0.1% formic acid-acetonitrile aqueous solution (acetonitrile content 84%). The liquid chromatography column (0.15mm*150mm, RP-C18, Column Technology Inc.) was equilibrated with 95% Solution A. Samples were loaded via an autosampler onto a Zorbax 300SB-C18 peptide traps (Agilent Technologies, Wilmington, DE) and then separated by the liquid chromatography column. The relevant liquid gradient settings were as follows: 0 min–50 min, linear gradient of Solution B from 4% to 50%; 50 min–54 min, linear gradient of Solution B from 50% to 100%; 54 min–60 min, Solution B maintained at 100%.

[0102] 2.3 Mass Spectrometry Conditions: The enzymatic digests were separated by capillary high-performance liquid chromatography (HPLC) and then analyzed by mass spectrometry using a Q Exactive HF-X mass spectrometer (Thermo Fisher). Analysis time: 60 min. Detection mode: positive ion. The mass-charge ratio of the peptide and peptide fragments was collected using the following method: 10 fragment spectra were acquired after each full scan (MS² scan).

[0103] 2.4 Data Analysis: The raw mass spectrometry test files were retrieved from the UniProt database using MaxQuant 1.5.5.1 software to obtain the protein identification and quantification results.

[0104] Results: By comparing the fragment ion information acquired by mass spectrometry with the UniProt database and a self-built peptide library, and by searching in MaxQuant software, the results showed that the four characteristic peptides shown in SEQ ID NO: 1 to SEQ ID NO: 4 were detected in high abundance in the enzymatic digest. Specific mass spectrometry identification parameters are shown in Table 1 and... Figure 2 As shown.

[0105] Table 1: LC-MS / MS identification results of characteristic peptides in the bird's nest peptide composition described in this application

[0106]

[0107] As shown in Table 1 and Figure 2 As shown, the measured mass-to-charge ratio ([M + H] +The deviations from the theoretical calculations were all less than 0.05 Da, and the fragment ion fingerprints (MS2) of each peptide completely matched the theoretical sequences. The differences in retention time were consistent with the hydrophobicity of each peptide (e.g., QLLL, rich in leucine L, eluted later, while PSGSF, containing polar amino acids, eluted earlier). These data fully demonstrate that the enzymatic hydrolysis and purification process described in this application can effectively release and enrich the four specific bioactive short peptides mentioned above.

[0108] Example 5: Anti-aging evaluation of bird's nest peptide composition in a zebrafish aging model

[0109] This embodiment uses the bird's nest peptide composition prepared in Example 1 for activity testing.

[0110] 5.1 Model Establishment and Safety Evaluation: In zebrafish embryo experiments, the survival rate of zebrafish embryos affects the experimental results. Therefore, it is necessary to determine the safe concentration of the experimental substances in zebrafish embryos beforehand through toxicity experiments. The experimentally determined safe concentration of the bird's nest peptide composition in zebrafish embryos was 50-250 μg / mL. In wild-caught AB strain zebrafish embryos, the mortality rate was low, their condition was good, and no deformities were observed. When the final concentration of the bird's nest peptide composition in the zebrafish culture system was 500 μg / mL, the mortality rate was high, and embryotoxic symptoms such as pericardial edema, trunk curvature, unresponsiveness to mechanical stimulation, and unclear muscle texture appeared. These results indicate that the bird's nest peptide composition is non-toxic to zebrafish embryos within the concentration range of 50-250 μg / mL, representing a safe concentration for zebrafish embryo experiments.

[0111] This experiment investigated the effect of different concentrations of H2O2 on zebrafish embryo survival. By detecting zebrafish embryo survival rates, the H2O2 concentration for zebrafish embryo senescence was selected, significantly reducing embryonic aging and thus determining the optimal H2O2 concentration for establishing a zebrafish embryo model. Zebrafish embryos were induced using different concentrations of H2O2, and embryo survival rates were observed after incubation at 28.5 ℃ for 48 h. Figure 3 It can be seen that no zebrafish embryos died in the blank control group and the 1, 2, 4, and 6 mmol / L H2O2 groups, with a survival rate of 100%. The survival rate of zebrafish embryos in the 8 mmol / L H2O2 group was 90% ± 4.08%, which was significantly lower than that in the blank control group (P < 0.05). Therefore, the 8 mmol / L H2O2 group was selected to establish a zebrafish embryo aging model.

[0112] 5.2 Effect of bird's nest peptide composition on the activity of SA-β-galactosidase (a marker of aging) induced by H2O2 in aging zebrafish embryos:

[0113] The study included a blank control group (0 mmol / L H2O2), a model group (8 mmol / L H2O2), and a bird's nest peptide group (bird's nest peptide composition + 8 mmol / L H2O2). The concentrations of the bird's nest peptide composition were 50 μg / mL, 100 μg / mL, and 250 μg / mL, respectively, and the results were repeated 3 times.

[0114] Each group consisted of 150 embryos, cultured in 6-well plates with 3 parallel wells (50 embryos per well). After drug treatment, the H2O2-treated zebrafish were washed three times with embryo culture medium and transferred to 1.5 mL centrifuge tubes. Pre-cooled PBS was added, and the mixture was sonicated. The homogenate was centrifuged at 4 ℃ and 10000 rpm / min for 10 min. The supernatant was collected, and the SA-β-galactosidase content in the zebrafish embryos was determined according to the SA-β-galactosidase manufacturer's instructions. The results are as follows: Figure 4 As shown, compared with the blank control group, the SA-β-galactosidase activity in the model group of zebrafish embryos was significantly increased (p<0.01), reaching 118% of the blank control group. After intervention with different concentrations of bird's nest peptide composition, the SA-β-galactosidase activity in the experimental group showed a concentration-dependent decreasing trend. Specifically, the enzyme activities in the 50 μg / mL, 100 μg / mL, and 250 μg / mL concentration groups were significantly reduced by 7.19% (p<0.05), 14.08% (p<0.01), and 18.47% (p<0.01) compared with the model group, respectively. This finding suggests that the bird's nest peptide composition can significantly reduce H2O2-induced oxidative damage by regulating oxidative stress-related biomarkers, and its mechanism of action may be closely related to its antioxidant properties such as scavenging free radicals and maintaining intracellular redox balance.

[0115] 5.3 Effect of bird's nest peptide composition on the activity of malondialdehyde (MDA) (lipid peroxidation product) induced by H2O2 in aging zebrafish embryos: A blank control group (0 mmol / L H2O2), a model group (8 mmol / L H2O2), and a bird's nest peptide group (bird's nest peptide composition + 8 mmol / L H2O2) were set up, with bird's nest peptide composition concentrations of 50 μg / mL, 100 μg / mL, and 250 μg / mL, respectively, and the results were repeated 3 times.

[0116] Each group consisted of 150 embryos, which were cultured in 6-well plates with 3 parallel wells, i.e., 50 embryos per well. After drug treatment, the H2O2-treated zebrafish were washed three times with embryo culture medium and then transferred to 1.5 mL centrifuge tubes. Pre-cooled PBS was added, and the mixture was homogenized using an ultrasonic homogenizer. The homogenate was centrifuged at 4℃ and 10000 rpm / min for 10 min. The supernatant was collected, and the MDA content in various zebrafish embryos was determined according to the MDA instructions.

[0117] The results are as follows Figure 5 As shown, compared with the blank control group, the malondialdehyde (MDA) content in zebrafish embryos in the model group was significantly increased (p<0.01), reaching 125% of that in the blank control group, indicating that H2O2 treatment successfully induced lipid peroxidation damage. After intervention with different concentrations of bird's nest peptide composition, the MDA content in each concentration group showed a decreasing trend compared with the model group. Among them, the MDA levels in the 50 μg / mL, 100 μg / mL, and 250 μg / mL bird's nest peptide composition treatment groups decreased by 3.77%, 6.40%, and 18.87%, respectively (p<0.01). As an end product of lipid peroxidation, the decrease in MDA content suggests that the bird's nest peptide composition may significantly reduce the damage to cell membrane structure caused by oxidative stress through pathways such as neutralizing free radicals and blocking the lipid peroxidation chain reaction, thereby maintaining cell membrane integrity and delaying the oxidative stress-related cellular senescence process.

[0118] 5.4 Effect of bird's nest peptide composition on superoxide dismutase (SOD) activity in H2O2-induced aging zebrafish embryos: A blank control group (0 mmol / L H2O2), a model group (8 mmol / L H2O2), and a bird's nest peptide group (bird's nest peptide composition + 8 mmol / L H2O2) were set up, with the concentrations of bird's nest peptide composition being 50 μg / mL, 100 μg / mL, and 250 μg / mL, respectively, and the experiment was repeated 3 times.

[0119] Each group consisted of 150 embryos, which were cultured in 6-well plates with 3 parallel wells (50 embryos per well). After drug treatment, the H2O2-treated zebrafish were washed three times with embryo culture medium and then transferred to 1.5 mL centrifuge tubes. Pre-cooled PBS was added, and the cells were homogenized using an ultrasonic homogenizer. The homogenate was centrifuged at 4°C and 10,000 rpm for 10 min. The supernatant was collected, and the SOD content in various zebrafish embryos was determined according to the SOD instructions.

[0120] The results are as follows Figure 6As shown, after H2O2 treatment, the superoxide dismutase (SOD) content in zebrafish embryos in the model group was significantly lower than that in the blank control group (p<0.01), reaching only 80.40% of that in the blank group, suggesting that oxidative stress leads to impaired function of the endogenous antioxidant defense system. After intervention with different concentrations of bird's nest peptide composition, the SOD activity in each experimental group showed a concentration-dependent recovery trend. Specifically, the SOD content in the 50 μg / mL, 100 μg / mL, and 250 μg / mL bird's nest peptide groups was significantly increased by 12.01% (p<0.05), 29.51% (p<0.01), and 43.25% (p<0.01), respectively, compared to the model group. Therefore, it is speculated that the bird's nest peptide composition effectively delays the H2O2-induced cellular senescence process by targeting and strengthening the SOD-mediated antioxidant defense system, providing direct experimental evidence for its anti-aging mechanism.

[0121] 5.5 Effect of bird's nest peptide composition on reactive oxygen species (ROS) levels induced by H2O2 in aging zebrafish embryos: A blank control group (0 mmol / L H2O2), a model group (8 mmol / L H2O2), and a bird's nest peptide group (bird's nest peptide composition + 8 mmol / L H2O2) were set up, with the concentrations of bird's nest peptide composition being 50 μg / mL, 100 μg / mL, and 250 μg / mL, respectively, and the results were repeated 3 times.

[0122] Each group consisted of 150 embryos, which were cultured in 6-well plates with 3 parallel wells (50 embryos per well). After drug treatment, the H2O2-treated zebrafish were washed three times with embryo culture medium and then transferred to 1.5 mL centrifuge tubes. Pre-cooled PBS was added, and the cells were homogenized using an ultrasonic homogenizer. The homogenate was centrifuged at 4°C and 10,000 rpm for 10 min. The supernatant was collected, and the ROS content in various zebrafish embryos was determined according to the ROS instructions.

[0123] The results were obtained by detecting the DCFH-DA fluorescent probe. Figure 7 As shown, after H2O2 induction, the reactive oxygen species (ROS) level in zebrafish embryos in the model group was significantly higher than that in the blank control group (p<0.01), reaching 157% of that in the blank group, indicating that oxidative stress leads to excessive accumulation of free radicals in cells. After intervention with different concentrations of bird's nest peptide composition, the ROS levels in each experimental group showed a significant dose-dependent inhibitory effect. Specifically, the ROS levels in the 50 μg / mL, 100 μg / mL, and 250 μg / mL bird's nest peptide groups were reduced by 42.13% (p<0.01), 81.78% (p<0.01), and 89.26% (p<0.01) respectively compared with the model group, suggesting that the bird's nest peptide composition can almost completely block the H2O2-induced ROS burst at higher doses.

[0124] 5.6 Effects of bird's nest peptide composition on the expression of senescence genes in H2O2-induced aging zebrafish embryos:

[0125] Sirt-1, p53, and p21 play crucial roles in the aging pathway. Sirt-1, as a deacetylase, regulates aging by influencing cellular metabolism, gene expression, and DNA repair through the regulation of the acetylation states of various proteins. p53, a tumor suppressor protein, is activated under stress, inducing cell cycle arrest or apoptosis to prevent malignant transformation. Simultaneously, p53 also participates in aging regulation; increased p53 activity promotes cellular senescence. p21, a downstream target gene of p53, encodes a protein that inhibits cell cycle progression, thus participating in aging regulation. In the aging pathway, Sirt-1 can influence p53 activity through deacetylation, thereby regulating p21 expression. Conversely, p53 can also regulate Sirt-1 activity through feedback mechanisms. This complex interaction creates a sophisticated regulatory network among Sirt-1, p53, and p21 in the aging pathway, collectively influencing the cellular aging process.

[0126] The study included a blank control group (0 mmol / L H2O2), a model group (8 mmol / L H2O2), and a bird's nest peptide group (bird's nest peptide composition + 8 mmol / L H2O2). The concentrations of the bird's nest peptide composition were 50 μg / mL, 100 μg / mL, and 250 μg / mL, respectively, and the results were repeated 3 times.

[0127] Each group consisted of 150 embryos, cultured in 6-well plates with 3 parallel wells (50 embryos per well). After drug treatment, the H2O2-treated zebrafish were washed three times with embryo culture medium and then transferred to 1.5 mL centrifuge tubes to remove excess water. Total RNA was extracted using RNA Simple. cDNA was synthesized from each sample using the reverse transcription kit according to the instructions. Detection was performed using quantitative real-time PCR. Primers for the housekeeping gene (GADPH) and target genes (Sirt-1, P53, P21) are shown in Table 2. Two... -△△CT The results were calculated using one-way ANOVA statistical analysis. p < 0.05 indicated a significant difference, and p < 0.01 indicated a highly significant difference.

[0128] Table 2

[0129]

[0130] (4) Reaction system and procedure: The SYBR Green method was used and performed on a QuantStudio real-time PCR instrument. Reaction conditions: 95℃ pre-denaturation for 30 seconds; 95℃ denaturation for 5 seconds, 60℃ annealing / extension for 30 seconds, for a total of 40 cycles.

[0131] (5) Data analysis: using 2 (-ΔΔCt) The relative expression levels of genes were calculated using a method with GAPDH as an internal reference gene.

[0132] The results are as follows Figure 8 As shown, the expression level of Sirt-1 in zebrafish embryos in the model group was significantly lower than that in the blank control group (p<0.01). Compared with the model group, Sirt-1 expression was significantly increased at concentrations of 50 μg / mL, 100 μg / mL, and 250 μg / mL of the bird's nest peptide composition (p<0.01), with increases of 62.7%, 77.8%, and 83.9%, respectively. This indicates that the bird's nest peptide composition can promote the expression level of Sirt-1 gene in zebrafish embryos induced by H2O2.

[0133] like Figure 9 and Figure 10 As shown, the expression of p53 and p21 genes in the model group was significantly upregulated (p<0.01). Intervention with the bird's nest peptide composition significantly inhibited the expression of these two genes (p<0.01).

[0134] The bird's nest peptide composition prepared in Example 1 can effectively activate the expression of the longevity gene SIRT1 and inhibit the downstream aging-related pathway gene p53 / p21, while comprehensively improving oxidative stress indicators, thus demonstrating its clear anti-aging efficacy at the whole animal model level.

[0135] Comparative Example 1:

[0136] The only difference between this comparative example and Example 1 is that in step (2), only alkaline protease (4000 U / g) is added, and trypsin is not added. The remaining steps are the same as in Example 1 to prepare the bird's nest peptide composition.

[0137] Comparative Example 2:

[0138] The only difference between this comparative example and Example 1 is that the intermediate powder obtained by spray drying in step (4) is used as the final product, and the HPLC separation and purification in step (5) is not performed. The remaining steps are the same as in Example 1 to prepare the bird's nest peptide composition.

[0139] Comparative Example 3:

[0140] The only difference between this comparative example and Example 1 is that the enzymatic hydrolysis temperature in step (2) is set to 70°C, while the other steps are the same as in Example 1, to prepare the bird's nest peptide composition.

[0141] Comprehensive performance comparison analysis

[0142] The products of Examples 1-3 (bird's nest peptide compositions) were compared with those of the internal comparative examples 1-3 and three commercially available bird's nest peptide composition products (A, B, C) in terms of physicochemical properties and core activity indicators (SOD and SIRT1 activation capacity). The results are shown in Table 3.

[0143] Table 3: Comprehensive Performance Comparison of Embodiments, Comparative Examples, and Commercially Available Products in this Application

[0144]

[0145] Note: The relative expression data of SIRT1 are based on the test results of a zebrafish model at a drug concentration of 250 μg / mL.

[0146] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of this application. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0147] Although embodiments of this application have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting this application. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of this application.

Claims

1. A bird's nest peptide composition that activates the longevity gene SIRT1, characterized in that, The bird's nest peptide composition comprises the four peptide segments shown in SEQ ID NO: 1 to SEQ ID NO: 4; The bird's nest peptide composition is prepared by the following method: (1) pre-treating the bird's nest raw material to obtain a bird's nest homogenate; (2) The bird's nest homogenate is subjected to enzymatic hydrolysis to obtain bird's nest hydrolysate; (3) The enzymatic hydrolysate of the bird's nest is concentrated by membrane and dried to obtain intermediate peptides with a molecular weight of less than 3 kDa; (4) The intermediate peptide is separated and purified, and the components with a molecular weight of 100-500 Da are collected to obtain the bird's nest peptide composition; In step (2), the enzymatic hydrolysis is performed at a temperature of 50-60℃ for 1-4 hours; the enzyme used in the enzymatic hydrolysis is a complex enzyme of alkaline protease and trypsin, and the total amount of the complex enzyme added is 3000-7000 U / g bird's nest protein. In step (4), the separation and purification are achieved by using a C18 column, with an aqueous solution containing 0.05%-0.15% trifluoroacetic acid as mobile phase A and an acetonitrile solution containing 0.05%-0.15% trifluoroacetic acid as mobile phase B, and gradient elution is performed. The gradient elution program is as follows: the proportion of mobile phase B is increased from 20% to 40% within 15 minutes, and then the proportion of mobile phase B is increased from 40% to 80% within 2 minutes.

2. The bird's nest peptide composition according to claim 1, characterized in that, After enzymatic hydrolysis, sterilization is carried out at 100°C for 15-25 minutes.

3. The bird's nest peptide composition according to claim 1, characterized in that, In step (3), the drying is achieved under the conditions of an inlet air temperature of 160-180℃ and an outlet air temperature greater than 90℃; and / or, the drying is spray drying.

4. A method for preparing the bird's nest peptide composition according to any one of claims 1 to 3, characterized in that, include: (1) Pre-process the bird's nest raw materials to obtain a homogenized bird's nest slurry; (2) The bird's nest homogenate is subjected to enzymatic hydrolysis to obtain bird's nest hydrolysate; (3) The enzymatic hydrolysate of the bird's nest is concentrated by membrane and dried to obtain intermediate peptides with a molecular weight of less than 3 kDa; (4) The intermediate peptide is separated and purified, and the components with a molecular weight of 100-500 Da are collected to obtain the bird's nest peptide composition; In step (2), the enzymatic hydrolysis is performed at a temperature of 50-60℃ for 1-4 hours; the enzyme used in the enzymatic hydrolysis is a complex enzyme of alkaline protease and trypsin, and the total amount of the complex enzyme added is 3000-7000 U / g bird's nest protein. In step (4), the separation and purification are achieved by using a C18 chromatographic column, with an aqueous solution containing 0.05%-0.15% trifluoroacetic acid as mobile phase A and an acetonitrile solution containing 0.05%-0.15% trifluoroacetic acid as mobile phase B, and gradient elution is performed. The gradient elution procedure is as follows: the proportion of mobile phase B is increased from 20% to 40% within 15 minutes, and then the proportion of mobile phase B is increased from 40% to 80% within 2 minutes.

5. The method according to claim 4, characterized in that, After enzymatic hydrolysis, sterilization is carried out at 100°C for 15-25 minutes.

6. The method according to claim 4, characterized in that, In step (3), the drying is achieved under the conditions of an inlet air temperature of 160-180℃ and an outlet air temperature greater than 90℃; and / or, the drying is spray drying.

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