Edible bird's nest peptide for improving mitochondrial function of ovarian granulosa cells, preparation method and application thereof

Abstract

The present application relates to the technical field of bird's nest deep processing, and discloses a bird's nest peptide for improving mitochondrial function of ovary granulosa cells, a preparation method and application thereof. In the bird's nest peptide, the proportion of combined sialic acid in total sialic acid is more than 80%, and the proportion of components with relative molecular mass less than 10 kDa is more than 80%. The bird's nest peptide is prepared from dried bird's nest through ultramicro-pulverization, vibration soaking and loosening, non-enzymatic and non-acid and alkali hydrolysis, multi-layer filtration, centrifugal separation and drying. The bird's nest peptide can improve ATP level and mitochondrial membrane potential of ovary granulosa cells and reduce ROS level.

Description

Technical Field

[0001] This invention relates to the field of bird's nest deep processing technology, and in particular to bird's nest peptides that improve mitochondrial function of ovarian granulosa cells, their preparation methods, and their applications. Background Technology

[0002] Bird's nest contains glycoproteins, sialic acid, and various amino acids, making it a commonly used animal-derived natural raw material in food and nutritional health products. In existing deep processing technologies for bird's nest, bird's nest peptides or extracts are typically obtained through enzymatic hydrolysis, digestion, acid hydrolysis, or heat treatment. For example, CN110229857A discloses a method for preparing bird's nest peptides and its depolymerization coupled with enzymatic hydrolysis, which involves soaking, denaturation, pulverization, pH adjustment, protease hydrolysis, enzyme inactivation, and filtration. CN108651994A discloses a novel bird's nest extract, its preparation method, and its application, which releases sialic acid from bird's nest and digests large bird's nest protein molecules into peptides through digestion. These technologies primarily focus on increasing the degradation degree of bird's nest protein, obtaining low-molecular-weight peptides, or increasing the content of free sialic acid, which can easily lead to structural damage to bird's nest peptides or an increase in the proportion of free small molecules. Existing technologies have not yet adequately addressed the issue of preserving the bound sialic acid glycoprotein peptide structure under non-enzymatic and non-acid-base conditions and using it to improve mitochondrial function in ovarian granulosa cells. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a bird's nest peptide with a high proportion of components with a relative molecular mass of less than 10 kDa, a high proportion of bound sialic acid retention, and the ability to improve the mitochondrial function of ovarian granulosa cells, as well as its preparation method and application.

[0004] To solve the above-mentioned technical problems, the first aspect of the present invention provides a bird's nest peptide that improves the mitochondrial function of ovarian granulosa cells. The bird's nest peptide is obtained by non-enzymatic hydrolysis and non-acid-base heating hydrolysis of dried bird's nest as raw material. The proportion of bound sialic acid in the bird's nest peptide is more than 80% of the total sialic acid, and the components with a relative molecular mass <10kDa account for more than 80% of the relative molecular mass distribution of the bird's nest peptide.

[0005] In some embodiments, the components with a relative molecular mass <10kDa account for more than 85% of the relative molecular mass distribution of the bird's nest peptide.

[0006] In some embodiments, the components with a relative molecular mass <10 kDa account for more than 90% of the relative molecular mass distribution of the bird's nest peptides. In some embodiments, the bound sialic acid accounts for more than 85% of the total sialic acid.

[0007] In some embodiments, the bound sialic acid accounts for more than 88% of the total sialic acid.

[0008] In some embodiments, the bound sialic acid accounts for more than 90% of the total sialic acid.

[0009] In some embodiments, the 3-10 kDa component of the bird's nest peptide accounts for 45-65% of the relative molecular mass distribution of the bird's nest peptide.

[0010] In some embodiments, the total sialic acid content of the bird's nest peptide is 9.0-12.0 wt%, the protein content is 60.0-66.0 wt%, and the total amino acid content is 52.0-57.0 wt%.

[0011] A second aspect of this invention provides a method for preparing the above-mentioned bird's nest peptide that improves mitochondrial function of ovarian granulosa cells, comprising the following steps:

[0012] S1. After removing impurities, cleaning and drying the dried bird's nest, it is then ultra-finely pulverized to obtain bird's nest powder.

[0013] S2. Mix the bird's nest powder with water, and soak it under mechanical vibration at 30-40℃. After standing and loosening, a bird's nest dispersion is obtained.

[0014] S3. The bird's nest dispersion is heated and hydrolyzed under stirring conditions to promote the hydrolysis and depolymerization of bird's nest glycoproteins, forming low molecular weight peptides and glycopeptide components, to obtain a heated hydrolysate of bird's nest; no protease, acid, alkali, organic solvent, preservative or exogenous sweetener is added during the heating and hydrolysis process;

[0015] S4. The bird's nest hydrolysate is heated and filtered through multiple layers to obtain filtrate;

[0016] S5. Centrifuge the filtrate and collect the supernatant;

[0017] S6. The supernatant is concentrated and dried to obtain bird's nest peptide that improves the mitochondrial function of ovarian granulosa cells.

[0018] In some embodiments, the dried bird's nest is edible dried bird's nest with a moisture content of not more than 12 wt% and a total sialic acid content of not less than 7.0 wt%.

[0019] In some embodiments, in step S1, the D90 particle size of the bird's nest powder is 40-80 μm.

[0020] In some embodiments, in step S2, the mass ratio of the bird's nest powder to water is 1:15-18.

[0021] In some embodiments, in step S2, the vibration frequency of the mechanical vibration soaking is 30-45Hz, and the vibration time is 60-100min.

[0022] In some embodiments, in step S3, the heating hydrolysis is either single-stage heating hydrolysis or segmented heating hydrolysis.

[0023] In some embodiments, the single-stage heating hydrolysis includes holding at 95-100°C for 4.0-6.0 hours.

[0024] In some embodiments, the segmented heating hydrolysis is a two-stage heating hydrolysis or a three-stage heating hydrolysis.

[0025] In some embodiments, the two-stage heating hydrolysis includes: first holding at 82-88℃ for 1.0-2.0h, and then holding at 95-100℃ for 3.0-5.0h.

[0026] In some embodiments, the three-stage heating hydrolysis includes: first holding at 72-82℃ for 0.5-1.5h, then holding at 86-94℃ for 1.0-3.0h, and finally holding at 95-100℃ for 2.0-4.0h.

[0027] In some embodiments, reflux condensation is used during the heating and hydrolysis process to reduce moisture loss.

[0028] In some embodiments, physical membrane fractionation is included after collecting the supernatant in step S5 and before concentrating and drying in step S6.

[0029] In some embodiments, the physical membrane fractionation process includes: firstly separating the supernatant using a membrane with a molecular weight cutoff of 10 kDa, collecting the permeate, and removing proteins, glycoprotein aggregates and macromolecular peptides with relatively large molecular weights to obtain bird's nest peptide components.

[0030] In some embodiments, the drying includes spray drying. The inlet air temperature of the spray dryer is 150-160°C, and the outlet air temperature is 72-78°C.

[0031] The third aspect of this invention provides the use of the bird's nest peptide that improves mitochondrial function of ovarian granulosa cells in the preparation of foods, health foods or nutritional compositions for improving mitochondrial function of ovarian granulosa cells.

[0032] In some embodiments, the improvement of ovarian granulosa cell mitochondrial function includes at least one of increasing ovarian granulosa cell ATP levels, increasing ovarian granulosa cell mitochondrial membrane potential, and decreasing ovarian granulosa cell ROS levels.

[0033] A fourth aspect of the present invention provides a food, health food, or nutritional composition for improving mitochondrial function of ovarian granulosa cells, comprising the bird's nest peptide for improving mitochondrial function of ovarian granulosa cells.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] This invention utilizes ultra-fine pulverization and mechanical vibration soaking to loosen the fibrous glycoprotein network of bird's nest, allowing it to fully absorb water and loosen its structure. Based on this, non-enzymatic hydrolysis and non-acid-base heating hydrolysis are employed to further promote the hydrolysis and depolymerization of bird's nest glycoproteins, forming low-molecular-weight peptides and glycopeptide components.

[0036] This invention utilizes single-stage enhanced heating hydrolysis, two-stage heating hydrolysis, or three-stage gradient heating hydrolysis to achieve a proportion of over 80% of the obtained bird's nest peptides with a relative molecular mass <10kDa. In particular, staged heating hydrolysis can increase the proportion of low-molecular-weight bird's nest peptides while reducing the excessive release of bound sialic acid from the glycopeptide structure.

[0037] The bird's nest peptides obtained by this invention not only have a high proportion of components <10kDa, but also a high proportion of bound sialic acid retention, which distinguishes them from conventional enzymatic hydrolysis and bird's nest processing routes that aim to obtain a large number of free small molecule peptides or release free sialic acid.

[0038] The bird's nest peptides obtained in this invention can improve cell viability, ATP levels, and mitochondrial membrane potential, and reduce ROS levels in an ovarian granulosa cell oxidative stress injury model, demonstrating their effect on improving mitochondrial function in ovarian granulosa cells. Further evidence from a desialylate treatment comparative study shows that, while maintaining a relatively constant overall molecular weight distribution, the disruption of the bound sialylate structure significantly reduces its effect on improving ATP levels, mitochondrial membrane potential, and ROS levels in ovarian granulosa cells. This indicates that the bound sialylate glycopeptide structure is a crucial active basis for the mitochondrial protective effect of the bird's nest peptides of this invention. Detailed Implementation

[0039] The following examples are used to further illustrate the present invention, but should not be construed as limiting the scope of protection of the present invention. Those skilled in the art can make conventional adjustments to the process parameters without departing from the concept of the present invention.

[0040] In the preparation process of this invention, no proteases, acids, alkalis, organic solvents, preservatives, sweeteners, or other exogenous chemical additives are added. The formation of bird's nest peptides mainly relies on ultrafine grinding, vibration soaking and loosening, and enhanced heating hydrolysis and depolymerization under non-enzymatic and non-acid-alkali conditions.

[0041] The bird's nest peptides referred to in this invention are bird's nest processing products obtained by non-enzymatic hydrolysis, non-acid-base heating hydrolysis and separation treatment using edible bird's nest as raw material. The components with a relative molecular mass of <10kDa account for more than 80%, and sialic acid mainly exists in the glycopeptide structure in a bound state.

[0042] Example 1

[0043] A method for preparing bird's nest peptides that improve mitochondrial function in ovarian granulosa cells includes the following steps:

[0044] Take 10.00 kg of dried bird's nest, remove feathers and visible impurities, rinse twice with purified water, and dry at 45℃ hot air until the moisture content is 7.0 wt%. Put the dried bird's nest into an ultra-micro pulverizer and pulverize it, controlling the temperature of the pulverizing chamber to not exceed 40℃ to obtain bird's nest micro powder. The D90 particle size of the bird's nest micro powder is 58 μm.

[0045] The bird's nest powder was mixed with purified water at a mass ratio of 1:16, and then mechanically vibrated and soaked at 34°C for 80 minutes at a vibration frequency of 38 Hz. After vibration, the mixture was allowed to stand for 40 minutes to obtain a bird's nest dispersion.

[0046] The bird's nest dispersion was transferred into a jacketed extraction tank equipped with a stirring and reflux condenser. The mixture was heated to 98°C at a stirring speed of 300 rpm and a heating rate of 1.5°C / min, and held at 98°C with stirring for 5.0 hours. This process allowed the fully water-absorbed and loosened bird's nest glycoproteins to undergo non-enzymatic, non-acid-base heating hydrolysis and depolymerization, yielding a heated bird's nest hydrolysate. During the heating process, reflux condensation was used to minimize water loss. No proteases, acids, alkalis, organic solvents, preservatives, or exogenous sweeteners were added during the entire heating hydrolysis process.

[0047] The heated hydrolysate of bird's nest is filtered sequentially through a 100-mesh sieve, a 200-mesh sieve, a 20μm filter element, and a 5μm filter element to remove insoluble coarse particles and residual impurities, thus obtaining the filtrate.

[0048] The filtrate was centrifuged twice: the first centrifugation was at 5000g for 15 min, and the second centrifugation was at 8000g for 10 min. The supernatant was collected. The supernatant was concentrated under vacuum at below 55°C until the solid content was 18.5 wt%. Then it was spray-dried at an inlet air temperature of 160°C and an outlet air temperature of 78°C to obtain 7.50 kg of bird's nest peptide powder.

[0049] The raw materials used in this embodiment are as follows:

[0050] Dried bird's nest, edible dried bird's nest, with a moisture content of 7.0 wt% and a total sialic acid content of not less than 7.0 wt%, derived from commercially available edible bird's nest raw materials.

[0051] Purified water, meeting the requirements for water used in food processing, originates from an on-site purified water system.

[0052] Example 2

[0053] A method for preparing bird's nest peptides that improve mitochondrial function in ovarian granulosa cells includes the following steps:

[0054] Take 10.00 kg of dried bird's nest, remove feathers and visible impurities, rinse twice with purified water, and dry at 45℃ hot air until the moisture content is 7.0 wt%. Put the dried bird's nest into an ultra-micro pulverizer and pulverize it, controlling the temperature of the pulverizing chamber to not exceed 40℃ to obtain bird's nest micro powder. The D90 particle size of the bird's nest micro powder is 58 μm.

[0055] The bird's nest powder was mixed with purified water at a mass ratio of 1:16, and then mechanically vibrated and soaked at 34°C for 80 minutes at a vibration frequency of 38 Hz. After vibration, the mixture was allowed to stand for 40 minutes to obtain a bird's nest dispersion.

[0056] The bird's nest dispersion was transferred to an extraction tank equipped with a stirring and reflux condenser. Two-stage heating hydrolysis was performed at a stirring speed of 260 r / min: first, the temperature was raised to 85℃ and held for 1.5 h to further expand and depolymerize the fully water-absorbed and loosened bird's nest glycoproteins; then, the temperature was raised to 98℃ and held for 4.0 h to further hydrolyze the bird's nest glycoproteins into low-molecular-weight peptides and glycopeptide components. The natural pH of the material was maintained during heating, and water loss was reduced through reflux condensation. No proteases, acids, alkalis, organic solvents, preservatives, or exogenous sweeteners were added, resulting in a heated hydrolysate of bird's nest.

[0057] The obtained bird's nest hydrolysate was filtered sequentially through a 100-mesh sieve, a 200-mesh sieve, a 30μm filter cartridge, a 10μm filter cartridge, and a 3μm filter cartridge to obtain the filtrate.

[0058] The filtrate was centrifuged three times: the first centrifugation was at 4000g for 15 min, the second centrifugation was at 7000g for 12 min, and the third centrifugation was at 10000g for 8 min. The supernatant was collected. The supernatant was concentrated under vacuum at below 52℃ until the solid content was 17.8wt%. Then it was spray-dried at an inlet air temperature of 155℃ and an outlet air temperature of 75℃ to obtain 7.18 kg of bird's nest peptide powder.

[0059] Compared to Example 1, this embodiment uses a two-stage heating hydrolysis method. First, the structure of bird's nest glycoproteins is expanded and depolymerized at a lower temperature, and then enhanced hydrolysis is carried out at a higher temperature. This is beneficial to increase the proportion of bird's nest peptides with a concentration of <10kDa, while reducing the excessive release of bound sialic acid caused by prolonged direct high-temperature treatment.

[0060] The raw materials used in this embodiment are as follows:

[0061] Dried bird's nest, edible dried bird's nest, with a moisture content of 7.0 wt% and a total sialic acid content of not less than 7.0 wt%, derived from commercially available edible bird's nest raw materials.

[0062] Purified water, meeting the requirements for water used in food processing, originates from an on-site purified water system.

[0063] Example 3

[0064] A method for preparing bird's nest peptides that improve mitochondrial function in ovarian granulosa cells includes the following steps:

[0065] Take 10.00 kg of dried bird's nest, remove feathers and visible impurities, rinse twice with purified water, and dry at 45℃ hot air until the moisture content is 7.0 wt%. Put the dried bird's nest into an ultra-micro pulverizer and pulverize it, controlling the temperature of the pulverizing chamber to not exceed 40℃ to obtain bird's nest micro powder. The D90 particle size of the bird's nest micro powder is 58 μm.

[0066] The bird's nest powder was mixed with purified water at a mass ratio of 1:16, and then mechanically vibrated and soaked at 34°C for 80 minutes at a vibration frequency of 38 Hz. After vibration, the mixture was allowed to stand for 40 minutes to obtain a bird's nest dispersion.

[0067] The bird's nest dispersion was transferred to an extraction tank equipped with a stirring and reflux condenser. A three-stage heating hydrolysis process was carried out at a stirring speed of 280 r / min: first, the temperature was raised to 78℃ and held for 1.0 h; then, it was raised to 90℃ and held for 2.0 h; finally, it was raised to 98℃ and held for 3.0 h, yielding the heated bird's nest hydrolysate. During the heating process, reflux condensation was used to reduce moisture loss. No proteases, acids, alkalis, organic solvents, preservatives, or exogenous sweeteners were added during the entire heating hydrolysis process.

[0068] The heated hydrolysate of bird's nest was filtered sequentially through a 100-mesh sieve, a 200-mesh sieve, a 20μm filter element, a 5μm filter element, and a 1μm filter element to obtain a filtrate. The filtrate was then centrifuged twice: the first centrifugation was performed at 6000g for 15min, and the second centrifugation was performed at 10000g for 10min. The supernatant was collected.

[0069] The supernatant was subjected to ultrafiltration fractionation using a membrane with a molecular weight cutoff of 10 kDa. The permeate was collected to obtain the bird's nest peptide component. The component was concentrated under vacuum at a temperature below 50°C to a solid content of 16.5 wt%, and then spray-dried at an inlet air temperature of 150°C and an outlet air temperature of 72°C to obtain 5.32 kg of bird's nest peptide powder enriched with bound sialic acid.

[0070] This embodiment improves the hydrolysis and depolymerization of bird's nest glycoproteins through three-stage gradient heating hydrolysis, removes macromolecular components larger than 10kDa through a 10kDa membrane, enriches bird's nest peptide components smaller than 10kDa, and maintains a high proportion of bound sialic acid.

[0071] The raw materials used in this embodiment are as follows:

[0072] Dried bird's nest, edible dried bird's nest, with a moisture content of 7.0 wt% and a total sialic acid content of not less than 7.0 wt%, derived from commercially available edible bird's nest raw materials.

[0073] Purified water, meeting the requirements for water used in food processing, originates from an on-site purified water system.

[0074] Comparative Example 1

[0075] A method for preparing enzymatically hydrolyzed bird's nest peptides includes the following steps:

[0076] Take 10.00 kg of dried bird's nest, remove feathers and visible impurities, rinse twice with purified water, and dry at 45℃ hot air until the moisture content is 7.0 wt%. Put the dried bird's nest into an ultra-micro pulverizer and pulverize it, controlling the temperature of the pulverizing chamber to not exceed 40℃ to obtain bird's nest micro powder. The D90 particle size of the bird's nest micro powder is 58 μm.

[0077] The bird's nest powder was mixed with purified water at a mass ratio of 1:16, and then mechanically vibrated and soaked at 34°C for 80 minutes at a vibration frequency of 38 Hz. After vibration, the mixture was allowed to stand for 40 minutes to obtain a bird's nest dispersion.

[0078] The bird's nest dispersion was heated to 55℃, the pH was adjusted to 7.0, and neutral protease was added at 0.6 wt% of the dry weight of the bird's nest. Enzymatic hydrolysis was carried out at a stirring speed of 300 r / min for 3.0 h. After the enzymatic hydrolysis was completed, the temperature was raised to 90℃ and kept at 10 min to inactivate the enzyme, thus obtaining the bird's nest enzymatic hydrolysate.

[0079] The enzymatic hydrolysate of bird's nest was filtered sequentially through a 100-mesh sieve, a 200-mesh sieve, a 20μm filter cartridge, and a 5μm filter cartridge. The resulting filtrate was centrifuged twice: the first centrifugation was at 5000g for 15min, and the second centrifugation was at 8000g for 10min. The supernatant was collected. The supernatant was then concentrated under vacuum at below 55℃ until the solid content was 18.0wt%, and then spray-dried at an inlet air temperature of 160℃ and an outlet air temperature of 78℃ to obtain 7.65kg of enzymatically hydrolyzed bird's nest peptide powder.

[0080] This comparative example illustrates the effects of conventional enzymatic hydrolysis on the structure of bird's nest peptides, retention of bound sialic acid, and molecular weight distribution.

[0081] The raw materials used in this comparative example are as follows:

[0082] Dried bird's nest, edible dried bird's nest, with a moisture content of 7.0 wt% and a total sialic acid content of not less than 7.0 wt%, derived from commercially available edible bird's nest raw materials.

[0083] Purified water, meeting the requirements for water used in food processing, originates from an on-site purified water system.

[0084] Neutral protease, a protease preparation for food processing, with an enzyme activity of 100,000 U / g.

[0085] Comparative Example 2

[0086] A method for preparing bird's nest powder extracted by ordinary hot water includes the following steps:

[0087] Take 10.00 kg of dried bird's nest, remove feathers and visible impurities, rinse twice with purified water, and dry at 45℃ hot air until the moisture content is 7.0 wt%. Put the dried bird's nest into a regular grinder and grind it, controlling the temperature of the grinding chamber to not exceed 40℃ to obtain bird's nest powder. The D90 particle size of the bird's nest powder is 210 μm.

[0088] Mix bird's nest powder with purified water at a mass ratio of 1:16, let it stand and soak for 60 minutes, without mechanical vibration to loosen it during soaking.

[0089] The soaked bird's nest powder dispersion was transferred into an extraction tank equipped with a stirring and reflux condensation device, heated to 98°C, and kept at this temperature for 5.0 hours under a stirring speed of 300 r / min. During the heating process, water loss was reduced by reflux condensation to obtain a common hot water extract.

[0090] The ordinary hot water extract was filtered sequentially through a 100-mesh sieve, a 200-mesh sieve, a 20μm filter cartridge, and a 5μm filter cartridge. The resulting filtrate was centrifuged twice: the first centrifugation was at 5000g for 15min, and the second centrifugation was at 8000g for 10min. The supernatant was collected. The supernatant was then concentrated under vacuum at below 55℃ until the solid content was 18.0wt%, and then spray-dried at an inlet air temperature of 160℃ and an outlet air temperature of 78℃ to obtain 6.48kg of ordinary hot water extracted bird's nest powder.

[0091] This comparative example is used to illustrate that, without the use of ultrafine grinding and mechanical vibration soaking and loosening, even with the same heating temperature and heating time as in Example 1, ordinary hot water treatment is still insufficient to obtain bird's nest peptide products with a high proportion of <10kDa components and good retention of bound sialic acid.

[0092] The raw materials used in this comparative example are as follows:

[0093] Dried bird's nest, edible dried bird's nest, with a moisture content of 7.0 wt% and a total sialic acid content of not less than 7.0 wt%, derived from commercially available edible bird's nest raw materials.

[0094] Purified water, meeting the requirements for water used in food processing, originates from an on-site purified water system.

[0095] Comparative Example 3

[0096] A method for preparing desialylated bird's nest peptides includes the following steps:

[0097] Take 1.00 kg of bird's nest peptide powder obtained in Example 3, add it to purified water, and prepare a bird's nest peptide solution with a mass concentration of 50 g / L. Stir at a stirring speed of 200 r / min for 30 min to fully dissolve it and obtain the bird's nest peptide solution.

[0098] The bird's nest peptide solution was adjusted to pH 5.5, heated to 37°C, and neuraminidase was added at 0.05 wt% of the bird's nest peptide mass. The mixture was stirred at 37°C for 4 hours to release bound sialic acid from the glycopeptide structure. After treatment, the system was heated to 90°C and kept at that temperature for 10 minutes to inactivate the enzyme, resulting in a desialic acid treated solution.

[0099] The desialyl treated solution was subjected to ultrafiltration desalting and removal of free small molecules using a membrane with a molecular weight cutoff of 1 kDa, and the retentate was collected. The retentate was concentrated under vacuum at a temperature below 50°C to a solid content of 16.0 wt%, and then spray-dried at an inlet air temperature of 150°C and an outlet air temperature of 72°C to obtain 0.82 kg of desialyl treated bird's nest peptide powder.

[0100] This comparative example illustrates the effect of bound sialic acid structure on the improvement of mitochondrial function in ovarian granulosa cells by bird's nest peptide.

[0101] The raw materials used in this comparative example are as follows:

[0102] The bird's nest peptide powder obtained in Example 3.

[0103] Neuraminidase: Neuraminidase for food or biochemical research, with an enzyme activity of 50 U / mg. Enzyme activity is defined as the amount of enzyme required to release 1 μmol of sialic acid per minute at 37°C and pH 5.5, which is derived from commercially available enzyme preparations.

[0104] Purified water, meeting the requirements for water used in food processing, originates from an on-site purified water system.

[0105] Test Example 1

[0106] Test of sialic acid, protein and amino acid composition in bird's nest peptides

[0107] Test samples: The test samples include the bird's nest peptides obtained in Examples 1-3 and the samples obtained in Comparative Examples 1-3.

[0108] Protein content was determined according to GB 5009.5-2025 "National Food Safety Standard - Determination of Protein in Food"; amino acid composition was determined according to GB 5009.124-2016 "National Food Safety Standard - Determination of Amino Acids in Food"; sialic acid was determined according to the first method of liquid chromatography-ultraviolet detection in GB 31614.1-2023 "National Food Safety Standard - Determination of Sialic Acid in Food".

[0109] The percentage of bound sialic acid is calculated using the following formula: Percentage of bound sialic acid = content of bound sialic acid / total sialic acid content × 100%.

[0110] Table 1. Results of sialic acid composition analysis in bird's nest peptides

[0111] Example 1 9.5 1.8 7.7 81.1 Example 2 10.2 1.3 8.9 87.3 Example 3 11.0 1.0 10.0 90.9 Comparative Example 1 8.7 3.2 5.5 63.2 Comparative Example 2 8.8 1.7 7.1 80.7 Comparative Example 3 3.6 0.8 2.8 77.8

[0112] Table 2. Results of protein and amino acid composition tests in bird's nest peptides

[0113] Example 1 61.8 53.7 27.4 Example 2 62.5 54.2 27.8 Example 3 64 55.1 28.3 Comparative Example 1 65.1 56.4 27.1 Comparative Example 2 52.8 45.6 26.8 Comparative Example 3 63.2 54.6 28.0

[0114] As shown in Tables 1-2, the proportion of bound sialic acid in the bird's nest peptides obtained in Examples 1-3 is all higher than 80%, with Example 3 reaching 90.9%. Compared with the enzymatically hydrolyzed bird's nest peptides in Comparative Example 1, the content of free sialic acid in Examples 1-3 is significantly reduced, while the proportion of bound sialic acid is significantly increased. This indicates that the non-enzymatic hydrolysis extraction process used in this invention can reduce the release of sialic acid from the glycoprotein peptide structure, which is beneficial for maintaining the bound sialic acid structure of the bird's nest peptides.

[0115] Compared with the ordinary hot water extraction sample of Comparative Example 2, the protein content, total amino acid content and total sialic acid content of Examples 1-3 were all higher, indicating that the ultrafine grinding, mechanical vibration soaking and loosening and heating hydrolysis processes are conducive to the full loosening, hydrolysis and depolymerization of the bird's nest glycoprotein network, which is not an effect that can be achieved by ordinary grinding and simple hot water treatment after static soaking.

[0116] Comparative Example 3 used the bird's nest peptide obtained in Example 3 as raw material and subjected it to desialylation treatment. Its protein content and total amino acid content were similar to those of Example 3, but the total sialylation content and bound sialylation content were significantly reduced. This indicates that the desialylation treatment mainly altered the sialylation-binding structure in the bird's nest peptide, without significantly changing its protein and amino acid composition. This comparative example can be used to further evaluate the effect of the bound sialylation structure on the mitochondrial protective effect of bird's nest peptide.

[0117] Test Example 2

[0118] Bird's Nest Peptide Molecular Weight Distribution Test

[0119] The relative molecular mass distribution of bird's nest peptides was determined by high-performance gel filtration chromatography, specifically referring to the determination principle and chromatographic method of peptide relative molecular mass distribution in Appendix A of GB / T22492-2008 "Soybean Peptide Powder", and the applicability of the method was confirmed in combination with the characteristics of bird's nest peptide samples.

[0120] Chromatographic conditions can be set as follows: TSKgel G2000 SWXL gel filtration column, acetonitrile-water-trifluoroacetic acid mobile phase, detection wavelength 220 nm, flow rate 0.5 mL / min, injection volume 20 μL. A calibration curve is established using peptide standards of known molecular weight, and the percentage area of ​​different molecular weight ranges is calculated.

[0121] The evaluation index refers to the requirements for the proportion of bird's nest peptides with a relative molecular mass of <10000 in T / CNFIA 172-2023 "Bird's Nest Peptides". The sum of the area percentages of components with a relative molecular mass of <1kDa, 1-3kDa and 3-10kDa is used as the proportion of components with a relative molecular mass of <10kDa.

[0122] Table 3. Relative molecular weight distribution of bird's nest peptides

[0123] Example 1 12.8 24.7 43.6 81.1 Example 2 10.6 23.8 51.4 85.8 Example 3 3.2 27.5 62.1 92.8 Comparative Example 1 40.2 31.8 18.9 90.9 Comparative Example 2 8.6 13.2 24.5 46.3 Comparative Example 3 4.0 28.0 59.9 91.9

[0124] As shown in Table 3, the proportions of the bird's nest peptides with a relative molecular mass <10kDa in Examples 1-3 were 81.1%, 85.8%, and 92.8%, respectively, all exceeding 80%. Specifically, Example 1 employed single-stage enhanced heating hydrolysis, yielding bird's nest peptides predominantly composed of <10kDa components; Example 2 used two-stage heating hydrolysis, further increasing the proportion of <10kDa components to 85.8%; and Example 3, through three-stage gradient heating hydrolysis and 10kDa membrane ultrafiltration fractionation, achieved a <10kDa component proportion of 92.8%, with 3-10kDa components accounting for 62.1%.

[0125] Comparative Example 1 was hydrolyzed with neutral protease, and the proportion of <10kDa components reached 90.9%. However, combined with Test Example 1, it can be seen that the content of free sialic acid is high and the proportion of bound sialic acid is low. This indicates that although conventional enzymatic hydrolysis can obtain more small molecule peptide components, it is easy to cause damage to the structure of bound sialic acid glycopeptides.

[0126] Comparative Example 2 was treated with ordinary hot water at the same heating temperature and time as Example 1. The proportion of the <10kDa component was only 46.3%, and the proportion of the component above 10kDa was relatively high. This indicates that under ordinary crushing and static soaking conditions, the bird's nest glycoprotein network cannot be fully loosened, and it is difficult to obtain bird's nest peptides with <10kDa components by simply heating.

[0127] The <10kDa component of Comparative Example 3 accounted for 91.9%, which is basically similar to that of Example 3. However, its bound sialic acid content was significantly reduced, indicating that Comparative Example 3 mainly changed the structure of bound sialic acid without significantly changing the overall molecular weight distribution of bird's nest peptide. Therefore, it can be used to evaluate the contribution of the bound sialic acid structure to the mitochondrial protective effect of bird's nest peptide.

[0128] Test Example 3

[0129] Effects of bird's nest peptides on mitochondrial function of ovarian granulosa cells

[0130] Human ovarian granulosa cells (KGN cells) were used as an in vitro model. KGN cells were purchased from the Cell Bank of the Chinese Academy of Sciences, with passage number P8-P15. Cells were cultured in DMEM / F12 medium containing 10 vol% fetal bovine serum and 1 vol% penicillin-streptomycin antibiotics at 37°C, 5% CO2, and saturated humidity.

[0131] The samples obtained in Examples 1-3 and Comparative Examples 1-3 were prepared into 10 mg / mL stock solutions using serum-free DMEM / F12 medium. After being fully dissolved, the solutions were filtered through a 0.22 μm sterile filter membrane for sterilization. Before use, the solutions were diluted to 100 μg / mL with complete culture medium.

[0132] Cell viability was assessed using 96-well plates, with KGN cells cultured at 5 × 10⁶ wells. 3 KGN cells were seeded at a density of 100 μL of complete culture medium per well and cultured for 24 h before treatment. ATP content, ROS levels, and mitochondrial membrane potential were measured using 6-well plates, with KGN cells seeded at a density of 2 × 10⁶ cells / well. 5 Inoculate at a density of 1 cell per well, add 2 mL of complete culture medium to each well, and incubate for 24 h before treatment.

[0133] During modeling, except for the control group, all cells in each group had their original culture medium discarded, were gently washed once with PBS, and then treated with serum-free culture medium containing 300 μmol / L H2O2 for 4 h to establish an oxidative stress injury model. After H2O2 treatment, the H2O2-containing culture medium was discarded, and the cells were gently washed once with PBS. The control group was cultured in fresh complete culture medium; the model group was cultured in fresh complete culture medium without the sample; and the cells in Example 1, Example 2, Example 3, Comparative Example 1, and Comparative Examples 2-3 were treated with fresh complete culture medium containing 100 μg / mL of the corresponding sample for 24 h.

[0134] After treatment, cell viability was assessed using the CCK-8 assay; intracellular ATP levels were detected using an ATP assay kit; intracellular ROS levels were detected using the DCFH-DA fluorescent probe assay; and mitochondrial membrane potential was detected using the JC-1 staining assay. Each group had six replicates, and the experiment was repeated three times. Results were considered as 100% for the control group, and ROS levels were also considered as 100% for the control group; higher ROS values ​​indicated higher levels of oxidative stress. The experimental groups are shown in Table 4 below.

[0135] Table 4 Experimental Groups

[0136] Blank group <![CDATA[No H₂O₂ added]]> Add fresh complete culture medium and continue culturing for 24 hours. Model group <![CDATA[Treatment with 300 μmol / L H₂O₂ for 4 h]]> <![CDATA[Discard the H2O2-containing medium, wash once with PBS, add fresh complete medium without samples and continue culturing for 24 h]]> Example 1 Group <![CDATA[Treatment with 300 μmol / L H₂O₂ for 4 h]]> <![CDATA[Discard the H2O2-containing medium, wash once with PBS, add fresh complete medium containing the sample of Example 1 at 100 μg / mL, and treat for 24 h]]> Example 2 group <![CDATA[Treatment with 300 μmol / L H₂O₂ for 4 h]]> <![CDATA[Discard the H₂O₂-containing medium, wash once with PBS, add fresh complete medium containing 100 μg / mL of the sample of Example 2, and treat for 24 h]]> Example 3 Group <![CDATA[Treatment with 300 μmol / L H₂O₂ for 4 h]]> <![CDATA[Discard the H₂O₂-containing medium, wash once with PBS, add fresh complete medium containing 100 μg / mL of the sample of Example 3, and treat for 24 h]]> Comparative Example 1 <![CDATA[Treatment with 300 μmol / L H₂O₂ for 4 h]]> <![CDATA[Discard the H2O2-containing medium, wash once with PBS, add fresh complete medium containing 100 μg / mL of the sample of Comparative Example 1, and treat for 24 h]]> Comparative Example 2 <![CDATA[Treatment with 300 μmol / L H₂O₂ for 4 h]]> <![CDATA[Discard the H2O2-containing medium, wash once with PBS, add fresh complete medium containing 100 μg / mL of the sample of Comparative Example 2, and treat for 24 h]]> Comparative Example 3 Groups <![CDATA[Treatment with 300 μmol / L H₂O₂ for 4 h]]> <![CDATA[Discard the H2O2-containing medium, wash once with PBS, add fresh complete medium containing 100 μg / mL of the sample of Comparative Example 3, and treat for 24 h]]>

[0137] Table 5. Effects of bird's nest peptides on mitochondrial function of ovarian granulosa cells

[0138] Blank group 100 100 100 100 Model group 61.8 55.6 218.4 52.3 Example 1 Group 80.4 78.2 146.5 75.8 Example 2 group 86.2 84.9 128.8 83.1 Example 3 Group 91.3 90.6 115.4 89.4 Comparative Example 1 72.3 68.1 170.4 66.9 Comparative Example 2 70.2 65.9 179.4 63.6 Comparative Example 3 Groups 73.0 68.8 168.7 67.0

[0139] Note: Cell viability, ATP level, and mitochondrial membrane potential are calculated with the blank group as 100%; ROS level is calculated with the blank group as 100%, and the higher the value, the stronger the oxidative stress.

[0140] Table 5 shows that after H2O2 treatment, cell viability, ATP levels, and mitochondrial membrane potential in the model group decreased significantly, while ROS levels increased significantly, indicating that ovarian granulosa cells in the model group experienced oxidative stress and mitochondrial dysfunction. Examples 1-3 all increased ATP levels and mitochondrial membrane potential in damaged cells and decreased ROS levels, with Example 3 showing the most significant effect. Compared with Comparative Example 1 (enzymatic hydrolysis of bird's nest peptides), Example 3 showed an increase in ATP level from 68.1% to 90.6%, an increase in mitochondrial membrane potential from 66.9% to 89.4%, and a decrease in ROS level from 170.4% to 115.4%. Compared with the ordinary hot water extract sample of Comparative Example 2, Example 3 also exhibited better mitochondrial protective effects.

[0141] Comparative Example 3, based on the bird's nest peptide obtained in Example 3, underwent desialylation treatment. Its molecular weight distribution and protein and amino acid composition were similar to those of Example 3, but the content of bound sialyl acid was significantly reduced. Compared to Example 3, Comparative Example 3 showed a significant decrease in its effects on restoring cell viability, ATP levels, and mitochondrial membrane potential, and its ROS reduction effect was also significantly weakened. This indicates that the bound sialyl acid structure in the bird's nest peptide is an important active structure for improving mitochondrial function in ovarian granulosa cells. These results demonstrate that the technical effects of the bird's nest peptide of this invention do not solely stem from ordinary peptide segments or amino acid composition, but are closely related to its retained bound sialyl glycopeptide structure.

[0142] The preferred embodiments of the present invention have been described in detail above. It should be understood that those skilled in the art can make numerous modifications and variations based on the concept of the present invention without creative effort. Therefore, all technical solutions that can be obtained by those skilled in the art based on the concept of the present invention through logical analysis, reasoning, or limited experimentation on the basis of existing technology should be within the scope of protection defined by the claims.

Claims

1. A bird's nest peptide that improves mitochondrial function in ovarian granulosa cells, characterized in that, The bird's nest peptide is obtained by non-enzymatic hydrolysis and non-acid-base heating hydrolysis of dried bird's nest as raw material; the proportion of bound sialic acid in the bird's nest peptide is more than 80% of the total sialic acid, and the components with a relative molecular mass <10kDa account for more than 80% of the relative molecular mass distribution of the bird's nest peptide.

2. The bird's nest peptide for improving mitochondrial function of ovarian granulosa cells as described in claim 1, characterized in that, The bound sialic acid accounts for more than 85% of the total sialic acid, the component with a relative molecular mass <10kDa accounts for more than 85% of the relative molecular mass distribution of the bird's nest peptide, and the component with a relative molecular mass of 3-10kDa accounts for 45-65% of the relative molecular mass distribution of the bird's nest peptide.

3. The bird's nest peptide for improving mitochondrial function of ovarian granulosa cells as described in claim 1, characterized in that, The total sialic acid content of the bird's nest peptide is 9.0-12.0 wt%, the protein content is 60.0-66.0 wt%, and the total amino acid content is 52.0-57.0 wt%.

4. A method for preparing the bird's nest peptide for improving mitochondrial function of ovarian granulosa cells as described in any one of claims 1-3, characterized in that, Includes the following steps: S1. After removing impurities, cleaning and drying the dried bird's nest, it is then ultra-finely pulverized to obtain bird's nest powder. S2. Mix the bird's nest powder with water, and soak it under mechanical vibration at 30-40℃. After standing and loosening, a bird's nest dispersion is obtained. S3. The bird's nest dispersion is heated and hydrolyzed under stirring conditions to obtain heated hydrolysate of bird's nest. No protease, acid, alkali, organic solvent, preservative and exogenous sweetener are added during the heating and hydrolysis process. S4. The bird's nest hydrolysate is heated and filtered through multiple layers to obtain filtrate; S5. Centrifuge the filtrate and collect the supernatant; S6. The supernatant is concentrated and dried to obtain bird's nest peptide that improves the mitochondrial function of ovarian granulosa cells.

5. The preparation method according to claim 4, characterized in that, The D90 particle size of the bird's nest powder is 40-80μm; the mass ratio of the bird's nest powder to water is 1:15-18; the vibration frequency of the mechanical vibration soaking is 30-45Hz, and the vibration time is 60-100min.

6. The preparation method according to claim 4, characterized in that, The heating hydrolysis is a segmented heating hydrolysis, which can be a two-stage heating hydrolysis or a three-stage heating hydrolysis; wherein, the two-stage heating hydrolysis includes: first holding at 82-88℃ for 1.0-2.0h, and then holding at 95-100℃ for 3.0-5.0h; the three-stage heating hydrolysis includes: first holding at 72-82℃ for 0.5-1.5h, then holding at 86-94℃ for 1.0-3.0h, and finally holding at 95-100℃ for 2.0-4.0h.

7. The preparation method according to claim 4, characterized in that, After collecting the supernatant in step S5 and before concentrating and drying in step S6, a physical membrane fractionation process is also included; the physical membrane fractionation process includes separating and collecting the bird's nest peptide components using a membrane with a molecular weight cutoff of 10 kDa.

8. The use of the bird's nest peptide as described in any one of claims 1-3 in the preparation of food, health food or nutritional composition for improving mitochondrial function of ovarian granulosa cells.

9. The application as described in claim 8, characterized in that, The improvement of ovarian granulosa cell mitochondrial function includes at least one of the following: increasing ATP levels in ovarian granulosa cells, increasing mitochondrial membrane potential in ovarian granulosa cells, and decreasing ROS levels in ovarian granulosa cells.

10. A food, health food, or nutritional composition for improving mitochondrial function in ovarian granulosa cells, characterized in that, Including the bird's nest peptide that improves mitochondrial function of ovarian granulosa cells as described in any one of claims 1-3.