Polypeptide synergistically enhanced cubilose collagen composite beverage with breast enlarging function and preparation method thereof

Abstract

The invention relates to the technical field of functional drinks, in particular to a polypeptide synergistically strengthened cubilose collagen composite drink with a breast enlarging function and a preparation method of the polypeptide synergistically strengthened cubilose collagen composite drink. The drink contains fish collagen tripeptide, elastin peptide, cubilose peptide, pueraria mirifica isoflavone, red clover isoflavone and saffron crocus extract. A lipidosome delivery system is adopted to entrap polypeptide to form a nano-scale compound, nano-emulsion entraps phytoestrogen, and berry polyphenol and lipidosome-polypeptide form a stable ternary compound. Through the multi-way synergistic effect that phytoestrogen activates a mammary gland estrogen receptor, polypeptide promotes connective tissue collagen synthesis and cubilose peptide activates a growth factor signal, mammary gland development is remarkably promoted, the breast enlarging effect is achieved, the bioavailability reaches 85% or above, and the retention rate of active ingredients stored at normal temperature for 24 months is larger than 90%.

Description

Technical Field

[0001] This invention relates to the field of functional beverage technology, and more specifically to a peptide-enhanced bird's nest collagen complex beverage with breast enhancement function and its preparation method, particularly to an oral beauty beverage that promotes breast development through the synergistic effect of phytoestrogens and peptides. Background Technology

[0002] With consumers' increasing demand for health and beauty, the oral beauty beverage market is experiencing rapid growth. Breast health and aesthetic appearance are important health concerns for women, and issues such as underdeveloped breasts and postpartum breast atrophy and sagging trouble many female consumers. Breast tissue is mainly composed of glandular tissue, adipose tissue, and connective tissue. Collagen and elastin, as core components of connective tissue, play a crucial role in maintaining the shape, elasticity, and firmness of the breasts. Factors such as aging, breastfeeding, and weight fluctuations can lead to collagen loss and elastic fiber breakage in the breasts, resulting in sagging and loosening.

[0003] Modern medical research shows that estrogen is a key hormone regulating breast development, stimulating the growth and development of mammary ducts and alveoli, and promoting the synthesis of collagen and elastic fibers in the mammary stroma. Phytoestrogens, such as isoflavones, have structures similar to endogenous estrogens. Similar to estradiol, it can bind to estrogen receptor α (ERα) and estrogen receptor β (ERβ) to exert estrogen-like effects without the side effects of synthetic estrogens. Bird's nest is rich in bioactive components such as sialic acid and enjoys a high reputation in the traditional health field. Modern research has confirmed its ability to promote the activation of the epidermal growth factor receptor (EGFR) signaling pathway. However, existing collagen-based bird's nest products generally suffer from low bioavailability, poor stability, and limited efficacy, especially lacking a systematic design for specific promotion of breast tissue.

[0004] Chinese patent CN107348294A discloses a bird's nest collagen solid beverage and its preparation method. The bird's nest collagen solid beverage provided by this patent comprises the following components and their weight percentages: 5%–30% bird's nest powder, 35%–45% collagen, 0.5%–1.5% water-soluble dietary fiber, 15%–28% sweetener, and 18%–22% guar gum. This invention uses an enzymatic method to extract collagen from fish skin. The solution undergoes degreasing and decolorization treatment, followed by enzymatic hydrolysis using a complex enzyme system of pepsin and papain. This invention produces a solid beverage with detoxifying, beautifying, lipid-lowering, and weight-loss effects. It can also increase the sialic acid content in breast milk from lactating women, promoting early intellectual development in infants.

[0005] However, the aforementioned existing technology still has the following shortcomings. First, the invention uses a solid beverage dosage form, requiring reconstitution before use, resulting in poor ready-to-drink properties, and the active ingredients may be destroyed during the reconstitution process. Second, the collagen used in this invention is ordinary macromolecular collagen, typically with a molecular weight exceeding 100,000 Da, requiring multiple stages of degradation in the body before absorption and utilization, with a bioavailability of only 15%–30%. Third, this invention uses only a single collagen as a functional protein source, failing to fully utilize the synergistic effects between different types of peptides, and also not addressing specific functional design for breast tissue. Furthermore, this invention lacks an effective antioxidant protection system and advanced encapsulation technology, leading to easy oxidative degradation of peptides during storage and use, resulting in insufficient product stability. Finally, the traditional enzymatic hydrolysis process used in this invention cannot achieve precise molecular weight control and nanoscale structure construction, limiting further improvement in product efficacy.

[0006] Chinese patent CN109831969A discloses a breast enhancement and beauty oral liquid and its preparation method. The oral liquid includes papaya extract, kudzu root extract, soy isoflavones, royal jelly freeze-dried powder, collagen peptides, vitamin E, potassium sorbate, and other ingredients. This invention achieves its breast enhancement effect through the combination of phytoestrogens and collagen. However, this patent also has significant shortcomings: first, it uses ordinary collagen peptides, and the molecular weight is not precisely controlled, resulting in limited absorption efficiency; second, it lacks an efficient delivery system, and the active ingredients are easily degraded in the gastrointestinal tract; third, it does not employ nanotechnology to improve bioavailability; fourth, the product stability protection measures are insufficient, and the activity of the effective ingredients is severely lost during storage; and fifth, the synergistic mechanism between the components is unclear, and there is a lack of systematic formulation optimization design.

[0007] Therefore, there is an urgent need to develop a novel bird's nest collagen complex beverage with high bioavailability, good stability, comprehensive efficacy, and targeted breast tissue promotion, as well as its preparation method, to meet consumers' demand for high-quality oral beauty and breast enhancement products. Summary of the Invention

[0008] To address the aforementioned problems in existing technologies, the present invention aims to provide a polypeptide-synergistic enhanced bird's nest collagen complex beverage with breast enhancement function and its preparation method. This beverage, by constructing a polypeptide synergistic system with a three-level molecular weight gradient, combined with a phytoestrogen nanoemulsion delivery system, and employing liposome encapsulation and polyphenol protection dual technologies, significantly improves the bioavailability, stability, and breast enhancement efficacy of the active ingredients, overcoming the shortcomings of existing technologies.

[0009] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0010] This invention provides a peptide-synergistic enhanced bird's nest collagen complex beverage with breast enhancement function, comprising the following components by weight percentage: fish collagen tripeptide powder 3.5%–5.0%, elastin peptide powder 1.5%–2.5%, bird's nest peptide powder 2.0%–3.5%, liposome delivery system 0.8–1.5 g / L, berry polyphenol protection system 2.0–4.0 g / L, and nanoemulsion system 1.5–2.5 g / L. The fish collagen tripeptide powder has a molecular weight of 280–500 Da, a degree of hydrolysis of 15%–18%, and a glycine-proline-hydroxyproline tripeptide sequence content greater than 30%. The elastin peptide powder has a molecular weight of 500–1500 Da and contains a valine-proline-glycine-valine-glycine repeating sequence. The bird's nest peptide powder has a molecular weight of 1000–3000 Da and a sialic acid content greater than 8%.

[0011] The liposome delivery system comprises phosphatidylcholine, cholesterol, tocopherol, and chitosan, encapsulating polypeptide components to form liposome-peptide complexes with a particle size of 80-120 nm. In the liposome delivery system, the mass ratio of phosphatidylcholine to cholesterol is 2.5:1 to 3.5:1, the polypeptide encapsulation efficiency of the liposome-peptide complex is 75%–82%, the polydispersity index is less than 0.25, and the zeta potential is -28 mV to -35 mV.

[0012] The berry polyphenol protection system comprises blueberry concentrate, cranberry concentrate, blood orange extract, and blackcurrant extract, which form a polyphenol-liposome-peptide ternary complex through non-covalent interactions with the liposome-peptide complex. The blueberry concentrate has an anthocyanin content greater than 25%, the cranberry concentrate has a proanthocyanidin A content greater than 15%, the blood orange extract has an anthocyanin content greater than 30%, and the blackcurrant extract has a total polyphenol content greater than 40%.

[0013] The nanoemulsion system consists of medium-chain triglycerides, polysorbate 80, Tween 20, and glyceryl monostearate, encapsulating Pueraria lobata extract, red clover extract, and saffron extract to form a nanoemulsion with an average particle size of 85 nm ± 15 nm. The Pueraria lobata extract contains more than 40% puerarin and more than 60% total isoflavones; the red clover extract contains more than 40% isoflavones; and the saffron extract contains more than 30% total crocin-I and crocin-II.

[0014] Furthermore, the beverage also includes 0.8-1.2 g / L of marine caviar extract powder, 0.15-0.25 g / L of γ-aminobutyric acid (GABA), 0.1-0.2 g / L of jujube seed extract, and 0.1-0.2 g / L of angelica extract. The fish collagen tripeptide powder, the elastin peptide powder, the bird's nest peptide powder, the marine caviar extract powder, the GABA, the jujube seed extract, and the angelica extract are cross-linked by transglutaminase catalysis to form a three-dimensional polypeptide network structure with a cross-linking degree of 12%–18%.

[0015] The polypeptide-enhanced bird's nest collagen complex beverage is a liquid beverage with a pH value of 3.8-4.2.

[0016] This invention also provides a method for preparing the above-mentioned peptide-synergistic enhanced bird's nest collagen complex beverage with breast enhancement function, comprising the following steps:

[0017] Step 1: Preparation of fish collagen tripeptide powder: Fish skin raw material was subjected to two-stage enzymatic hydrolysis using alkaline protease and neutral protease. The first stage was carried out at pH 9.0 and 50℃ for 4 hours, and the second stage at pH 7.0 and 55℃ for 3 hours, controlling the degree of hydrolysis to 15%–18%. The hydrolysate was filtered through a ceramic membrane and concentrated by nanofiltration to a solids content of 25%–30%, then freeze-dried under vacuum to obtain the fish collagen tripeptide powder. The alkaline protease had an activity of 200,000 U / g and an enzyme addition amount of 0.3%, while the neutral protease had an activity of 100,000 U / g and an enzyme addition amount of 0.2%.

[0018] Step 2, Preparation of elastin peptide powder: The elastin raw material was enzymatically hydrolyzed with trypsin at pH 8.0 and 40℃ for 5 hours. The hydrolysate was separated by ultrafiltration membrane, concentrated to a solid content of 25%, and then freeze-dried under vacuum to obtain the elastin peptide powder. The trypsin had an enzyme activity of 50,000 U / g, and the enzyme addition amount was 0.25%.

[0019] Step 3, Preparation of bird's nest peptide powder: Bird's nest raw materials were extracted using supercritical CO2 fluid at a pressure of 25 MPa and a temperature of 45°C for 2 hours. The extracted bird's nest residue was mixed with purified water at a mass ratio of 1:20. Enzymatic hydrolysis was performed using a combination of alkaline and neutral protease enzymes at pH 7.5 and a temperature of 55°C for 6 hours, controlling the degree of hydrolysis to 25%–30%. The hydrolysate was then centrifuged, concentrated by nanofiltration to a solid content of 20%–25%, and freeze-dried under vacuum to obtain the bird's nest peptide powder. The amount of alkaline protease added was 0.15%, and the amount of neutral protease added was 0.10%.

[0020] Step 4, Preparation of liposome-peptide complex: Phosphatidylcholine, cholesterol, tocopherol, and chitosan were dissolved in anhydrous ethanol-chloroform mixed solvent, and rotary evaporated to form a lipid film. A phosphate buffer solution containing the fish collagen tripeptide powder, the elastin peptide powder, and the bird's nest peptide powder was added for hydration. The mixture was ultrasonicated at 42 kHz and 400 W for 15 min, and then homogenized three times under high pressure at 600 bar to obtain the liposome-peptide complex. The mass ratio of phosphatidylcholine, cholesterol, tocopherol, and chitosan was 800:250:200:120, and the volume ratio of the anhydrous ethanol-chloroform mixed solvent was 1:3. The rotary evaporation was carried out at 50 °C and a vacuum of -0.09 MPa for 1 h.

[0021] Step 5: Preparation of the polyphenol-liposome-peptide ternary complex: Blueberry concentrate, cranberry concentrate, blood orange extract, and blackcurrant extract were dissolved in an acetate-sodium acetate buffer solution at pH 4.5. The liposome-peptide complex suspension was added dropwise to the berry polyphenol solution and stirred at 25°C for 2 hours. The mixture was then purified by gel filtration chromatography and dialysis to obtain the polyphenol-liposome-peptide ternary complex. The mass ratio of the blueberry concentrate, cranberry concentrate, blood orange extract, and blackcurrant extract was 1.2:0.7:0.6:0.5. The concentration of the acetate-sodium acetate buffer solution was 50 mM. The stirring speed was 300 rpm. The gel filtration chromatography used a Sephadex G-100 gel, and the dialysis was performed using a dialysis bag with a molecular weight cutoff of 12-14 kDa at 4°C for 24 hours in a phosphate buffer solution at pH 7.4.

[0022] Step 6, Preparation of nanoemulsion: Medium-chain triglycerides were mixed with Pueraria lobata extract, red clover extract, and saffron extract to form an oil phase. Polysorbate 80, Tween 20, and glyceryl monostearate were dissolved in purified water to form an aqueous phase. The aqueous phase was added dropwise to the oil phase using a reverse emulsification method. The mixture was ultrasonically treated for 15 min at a frequency of 42 kHz and a power of 500 W, and then homogenized under high pressure for 3 times at a pressure of 600 bar to obtain a nanoemulsion with an average particle size of 85 nm ± 15 nm.

[0023] Step 7, Enzymatic Crosslinking Treatment: The polyphenol-liposome-peptide ternary complex was mixed with the nanoemulsion, and marine caviar extract powder, γ-aminobutyric acid, jujube seed extract, and angelica extract were added. The pH was adjusted to 6.5-7.0, and transglutaminase was added. The reaction was carried out at 40℃ for 4 hours, controlling the degree of crosslinking to 12%-18%. The temperature was then raised to 85℃ and held for 15 minutes to inactivate the enzyme, yielding the enzymatically crosslinked product. The transglutaminase had an enzyme activity of 100 U / g and was added at 0.5% of the total protein mass. During the enzymatic crosslinking reaction, the degree of peptide polymerization was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis every 1 hour.

[0024] Step 8, Microencapsulation: Prepare a wall material solution containing maltodextrin, gum arabic, modified starch, and sodium alginate. Mix the solution with the enzymatic crosslinking product at a core-to-wall material mass ratio of 1:3 and emulsify. Process using spray drying technology, with an inlet air temperature of 155-165℃ and an outlet air temperature of 75-85℃, to obtain microcapsule powder with an average particle size of 80nm±25nm and an encapsulation rate greater than 88%. The maltodextrin has a glucose equivalent of 15-20, the modified starch is sodium octenyl succinate starch, and the mass ratio of maltodextrin, gum arabic, modified starch, and sodium alginate is 15:8:6:1. The atomization pressure of the spray drying is 3.0MPa, the feed rate is 600-800mL / h, and the atomizer speed is 20000rpm.

[0025] Step 9, final preparation: Sweetener, pH adjuster, and thickener are added sequentially to purified water, followed by the microcapsule powder. After two-stage homogenization, the mixture is sterilized by ultra-high temperature instantaneous sterilization at 138℃ for 4 seconds, and then filled under aseptic conditions to obtain the polypeptide-synergistic-enhanced bird's nest collagen complex beverage with breast enhancement function.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] First, this invention constructs a three-tiered molecular weight gradient polypeptide synergistic system composed of collagen tripeptide, elastin peptide, and bird's nest peptide, and innovatively combines it with a phytoestrogen nanodelivery system to achieve a multi-dimensional synergistic promoting effect on breast tissue. The small-molecule collagen tripeptide, with a molecular weight of 280-500 Da, can be directly absorbed into the bloodstream via the peptide transporter PepT1 in the intestine, rapidly reaching the breast connective tissue to provide direct raw materials for collagen synthesis in fibroblasts, stimulating the production of type I and type III collagen and enhancing the structural support of the breast. The medium-molecule elastin peptide, with a molecular weight of 500-1500 Da, has a characteristic valine-proline-glycine sequence that specifically stimulates fibroblasts to synthesize elastin, rebuilding the elastic fiber network of the breast and improving sagging and looseness. The large-molecule bird's nest peptide, with a molecular weight of 1000-3000 Da, is rich in sialic acid glycopeptide structure, which can activate the epidermal growth factor receptor (EGFR) signaling pathway, promoting the proliferation and differentiation of mammary duct epithelial cells, while being slowly released in the gastrointestinal tract, providing sustained immunomodulatory and nutritional effects. The three polypeptides with different molecular weights complement each other in terms of absorption rate, target site, and duration of efficacy, increasing bioavailability from 15%–30% in existing technologies to over 85%, thus achieving comprehensive promotion of mammary gland tissue and supporting tissue.

[0028] Secondly, this invention innovatively employs a nanoemulsion system to deliver phytoestrogens complexes, significantly improving the bioavailability and targeting of estrogen-like components to breast tissue. Pueraria lobata extract is rich in isoflavones such as puerarin and genistein, with puerarin content exceeding 40% and total isoflavone content exceeding 60%. These components can selectively bind to estrogen receptor β (ERβ) highly expressed in breast tissue, activating estrogen receptor-mediated gene transcription, upregulating the expression of breast development-related genes such as PR (progesterone receptor), IGF-1 (insulin-like growth factor 1), and VEGF (vascular endothelial growth factor), and promoting mammary duct branching and alveolar development. Red clover extract has an isoflavone content exceeding 40%, with its main components being genistein, daidzein, chickpea sprout A, and gentianin. It exhibits stronger estrogenic activity than soy isoflavones and can synergistically enhance the response of breast tissue to estrogen signals. The total content of crocin-I and crocin-II in saffron extract is greater than 30%, which not only promotes blood circulation and improves microcirculation in breast tissue, but also regulates the hypothalamus-pituitary-ovarian axis, optimizing the secretion rhythm of endogenous estrogen and progesterone, and creating a favorable endocrine environment for breast development. These lipid-soluble phytoestrogens are encapsulated in nanodroplets with a particle size of 85nm±15nm using nanoemulsification technology. This nanoscale gives the droplets an ultra-large specific surface area and excellent membrane permeability, increasing the bioavailability of phytoestrogens by more than 3 times compared to ordinary formulations. Furthermore, they can be absorbed through the lymphatic system, bypassing the first-pass effect of the liver, and increasing the effective concentration reaching breast tissue.

[0029] Third, this invention employs a liposome-nanoemulsion dual delivery system combined with polyphenol-peptide complex protection technology to achieve efficient delivery, stable protection, and synergistic effects of active ingredients. The liposomes are composed of biocompatible materials such as phosphatidylcholine and cholesterol, forming bilayer vesicles similar in structure to cell membranes, with a particle size controlled at 80-120 nm and an encapsulation efficiency of 75%–82%. This lipid bilayer structure protects peptides from degradation by gastric acid and digestive enzymes, and promotes peptides to cross the intestinal epithelial cell barrier and enter the bloodstream through mechanisms such as membrane fusion and endocytosis. The addition of chitosan forms a hydrophilic protective layer on the surface of the liposomes, prolonging in vivo circulation time and enhancing targeted enrichment in breast tissue. The construction of the polyphenol-peptide complex utilizes the multiple phenolic hydroxyl groups of polyphenolic compounds such as blueberry anthocyanins, cranberry proanthocyanidins, and blood orange anthocyanins to form stable complexes with the amino, carboxyl, and amide groups on the peptide chain through hydrogen bonds, electrostatic interactions, and hydrophobic interactions. This complex protects the active sites of the peptides from oxidative degradation through steric hindrance, reducing the rate of oxidative degradation by 65%, and maintaining over 90% retention of active ingredients after 24 months of storage at room temperature. Furthermore, the polyphenolic compounds themselves possess strong free radical scavenging capabilities, with a DPPH free radical scavenging rate exceeding 92%. This effectively eliminates reactive oxygen species generated by estrogen metabolism in breast tissue, protecting breast cell DNA from oxidative damage and reducing the risk of breast hyperplasia and fibrosis. This creates a safe synergistic effect with the peptides' breast-promoting effects.

[0030] Fourth, this invention employs transglutaminase-catalyzed peptide cross-linking to construct a three-dimensional network structure, and uses microencapsulation technology for composite protection, significantly improving product stability and in vivo sustained-release effect. Transglutaminase catalyzes an acyl transfer reaction between the γ-amide group of glutamine residues and the ε-amino group of lysine residues on the peptide chain, forming covalently cross-linked ε-glutamyl-lysine isopeptide bonds, connecting free peptide chains into a three-dimensional network structure. The degree of cross-linking is controlled within the range of 12% to 18%, ensuring both the structural stability of the peptide network and retaining sufficient free active sites for cell receptor recognition and binding. This enzymatic cross-linking is carried out under mild conditions, without damaging the secondary structure and biological activity of the peptide. The cross-linked peptide network is then microencapsulated using natural polysaccharides such as maltodextrin and gum arabic as wall materials, and a dense protective layer is formed through spray drying technology, achieving an encapsulation rate of over 88%, with the microcapsule particle size controlled at 80nm ± 25nm. Microencapsulation not only further improves the product's tolerance to environmental factors such as temperature, light, and oxygen, but also endows it with sustained-release properties, allowing the active ingredients to be gradually released in the digestive tract, prolonging the duration of action and reducing the frequency of administration. In vitro release experiments show that microencapsulated peptides release no more than 15% within 2 hours in simulated gastric juice and more than 85% continuously within 8 hours in simulated intestinal juice. This sustained-release mode is beneficial for maintaining the steady-state concentration of peptides and phytoestrogens in the blood, and continuously stimulating the growth and development of mammary tissue.

[0031] Fifth, this invention employs supercritical fluid. A combined extraction-enzymatic hydrolysis technique was used to prepare bird's nest peptides, increasing the sialic acid recovery rate by 35% compared to traditional methods, reaching over 92%. Supercritical fluid extraction was employed. The fluid is in a supercritical state under pressure of 25 MPa and temperature of 45 °C, exhibiting low viscosity like a gas and high solubility like a liquid. The carbonic acid formed when dissolved in water is weakly acidic. This weakly acidic environment selectively promotes the release of sialic acid from the glycoprotein structure of bird's nest, without causing the destruction and isomerization of sialic acid as strong acid hydrolysis would. The extraction temperature of 45℃ is much lower than the 95℃ of traditional water extraction, completely avoiding the thermal degradation of sialic acid. The extracted bird's nest residue is then hydrolyzed by a complex enzyme system of alkaline and neutral proteases, efficiently converting the protein into small molecule peptides with a molecular weight of 1000-3000 Da, with the degree of hydrolysis controlled at 25%-30%. The two-step process achieves segmented and optimized extraction of sialic acid and peptides, with a sialic acid content of more than 8% in the product, significantly higher than the 3%-5% of existing technologies. As an important component of gangliosides and glycoproteins, sialic acid not only promotes the development of the nervous system and immune regulation, but also promotes the differentiation and maturation of mammary epithelial cells in breast tissue, enhances the sensitivity of the mammary gland to prolactin, and provides nutritional support for postpartum lactation and secondary breast development.

[0032] Sixth, the formula of this invention also includes auxiliary ingredients such as marine caviar extract powder, γ-aminobutyric acid (GABA), jujube seed extract, and angelica extract, forming a complex nutritional system targeting female endocrine regulation and breast health. Marine caviar extract is rich in nucleic acids, amino acids, and minerals. Trace elements provide nutritional support for ovarian function, optimizing the synthesis and secretion of estrogen and progesterone. Gamma-aminobutyric acid (GABA), as an inhibitory neurotransmitter in the central nervous system, can regulate the function of the hypothalamus-pituitary axis, alleviate the inhibitory effect of stress on the endocrine system, and improve menstrual irregularities and breast hyperplasia caused by mental stress. Jujube seed extract is rich in jujube seed saponin A, which has a calming and sleep-improving effect; sufficient sleep is an important prerequisite for maintaining normal sex hormone secretion. Angelica extract contains more than 1% ferulic acid; Angelica has the effects of nourishing blood, promoting blood circulation, regulating menstruation, and relieving pain, which can improve blood circulation in breast tissue, promote the delivery of nutrients and hormones to breast tissue, and relieve discomfort such as premenstrual breast tenderness. These auxiliary components, in synergy with peptides and phytoestrogens, support healthy breast development and functional maintenance from multiple dimensions, including nutritional supply, neuroendocrine regulation, and improved blood circulation.

[0033] Seventh, this invention is a ready-to-drink liquid beverage, employing ultra-high temperature (UHT) sterilization and aseptic filling technology to achieve a long shelf life of 24 months at room temperature while maintaining excellent sensory quality and the stability of active ingredients. UHT sterilization, performed at 138°C for 4 seconds, achieves commercial sterility standards. Due to the extremely short processing time, the loss of activity of heat-sensitive peptides, phytoestrogens, and polyphenols is minimal, with tests showing an active ingredient retention rate of over 95%. The slightly acidic environment, controlled at a pH of 3.8-4.2, not only provides a refreshing taste but also inhibits microbial growth and Maillard reactions, contributing to product stability during storage. Compared to existing solid beverages that require preparation, this invention's liquid beverage is ready to drink upon opening, significantly improving consumer convenience and avoiding the adverse effects of water temperature and quality on active ingredients during preparation, ensuring the complete intake of beneficial components.

[0034] Eighth, the formulation design of this invention fully considers the physiological characteristics and developmental patterns of breast tissue, achieving a synergistic promotion effect across multiple targets, pathways, and levels. At the molecular level, phytoestrogens directly stimulate the proliferation of mammary epithelial cells and the development of mammary ducts through the estrogen receptor signaling pathway. Collagen tripeptides and elastin peptides provide structural protein synthesis raw materials for mammary connective tissue and stimulate fibroblast activity. Bird's nest peptides promote epithelial cell differentiation and maturation through the EGFR signaling pathway. These three signaling pathways mutually promote each other, forming a positive feedback regulatory network. At the tissue structure level, the synthesis of collagen and elastin enhances the support and elasticity of the breast, improving its shape and firmness. A moderate increase in adipose tissue enriches breast volume, and the development of glandular tissue perfects breast function. At the systemic regulatory level, the auxiliary components regulate the endocrine, nervous, and circulatory systems, creating a favorable internal environment and nutrient supply for breast development. This multi-dimensional synergistic mechanism makes the breast enhancement effect of this invention significantly superior to existing products that simply supplement collagen or use phytoestrogens.

[0035] In summary, this invention comprehensively overcomes the shortcomings of existing technologies by constructing a three-level polypeptide synergistic system, employing phytoestrogens for nanoemulsion delivery, utilizing liposome-peptide complex and polyphenol protection technology, applying enzymatic cross-linking-microencapsulation composite stabilization technology, and employing a supercritical extraction-enzymatic hydrolysis combined process. This significantly improves the product's bioavailability, stability, and breast enhancement efficacy, while ensuring product safety, providing an innovative technical solution for the high-end oral beauty and breast enhancement beverage market. Detailed Implementation

[0036] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0037] The main raw materials used in this invention and their sources are as follows: fish skin is selected from fresh deep-sea tilapia or cod skin; pig aortic blood vessels are purchased from Henan Shuanghui Industrial Group Co., Ltd.; white bird's nest is selected from first-grade bird's nest from Indonesia; blueberries, cranberries, blood oranges, and blackcurrants are all selected from frozen fresh fruits; soybean lecithin and phosphatidylcholine have a purity greater than 95%; cholesterol is purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., with a purity greater than 99%; tocopherol is purchased from Zhejiang Pharmaceutical Co., Ltd., in the form of vitamin E oil. Content 98%; Chitosan, degree of deacetylation greater than 90%; Medium-chain triglycerides, polysorbate 80, and Tween 20 were purchased from Kelun Pharmaceutical Co., Ltd.; Pueraria lobata raw material was purchased from Guangxi Wuzhou Zhongheng Group Co., Ltd.; Red clover raw material was purchased from a Chinese medicinal herb planting base in Tianshui City, Gansu Province; Saffron raw material was purchased from Tibet Tibetan Medicine Group Co., Ltd.; Transglutaminase was purchased from Ajinomoto Co., Ltd., Japan, with an enzyme activity of 100 U / g; Alkaline protease was purchased from Novozymes (China) Biotechnology Co., Ltd., under the trade name Alcalase. 2.4L FG, enzyme activity 200,000 U / g; neutral protease purchased from Novozymes (China) Biotechnology Co., Ltd., trade name Neutrase 0.8L, enzyme activity 100,000 U / g; trypsin purchased from Sigma-Aldrich, enzyme activity 50,000 U / g; maltodextrin, glucose equivalent DE18; gum arabic purchased from Inner Mongolia Alashan Left Banner Linyuan Natural Plant Development Co., Ltd.; sodium octenyl succinate starch purchased from Shandong Ruitai Starch Co., Ltd.; sodium alginate purchased from Qingdao Mingyue Seaweed Group Co., Ltd.; marine caviar extract powder purchased from Dalian; γ-aminobutyric acid, purity greater than 98%; jujube seed raw material purchased from Xingtai City, Hebei Province, Chinese medicinal herb planting base; angelica raw material purchased from Minxian County, Gansu Province, Chinese medicinal herb market; other conventional reagents were all food grade, purchased from Sinopharm Chemical Reagent Co., Ltd.

[0038] Example 1: This example describes the preparation of a peptide-enhanced bird's nest collagen complex beverage with breast-enhancing function, using the lower limit of the formula. In this example, the addition amount of fish collagen tripeptide powder is 3.5%, elastin peptide powder is 1.5%, and bird's nest peptide powder is 2.0%, reflecting the minimum addition range of the formula composition.

[0039] The compound beverage is prepared using the following steps. First, fish collagen tripeptide powder is prepared. 10 kg of fresh tilapia skin is selected, cleaned to remove surface mucus and impurities, and placed in a stainless steel reactor. 100 L of purified water is added, the pH is adjusted to 9.0, and the mixture is heated to 50°C. 30 g of alkaline protease is added for the first stage of enzymatic hydrolysis, and the reaction is maintained at 50°C and pH 9.0 for 4 hours to initially hydrolyze the large-molecule collagen into intermediate products with a molecular weight of approximately 5000-10000 Da. After the first stage of enzymatic hydrolysis is completed, the pH is adjusted to 7.0, 20 g of neutral protease is added, and the temperature is raised to 55°C for further enzymatic hydrolysis for 3 hours. During this process, samples are taken every 30 minutes, and the free amino content is determined using the trinitrobenzenesulfonic acid method to calculate the degree of hydrolysis. The reaction is stopped when the degree of hydrolysis reaches 15%. The temperature is immediately raised to 90°C and maintained for 10 minutes to inactivate the protease. After cooling to room temperature, the mixture is filtered through a ceramic membrane with a molecular weight cutoff of 1000 Da to remove large-molecule proteins and insoluble matter. The filtrate was concentrated using a nanofiltration membrane with a molecular weight cutoff of 500 Da until the solid content reached 25%. The concentrate was then transferred to a freeze dryer, with a pre-freezing temperature set at -45°C for 12 hours. Sublimation drying was then initiated, with the sublimation temperature gradually increased from -35°C to -25°C, maintaining a vacuum below 10 Pa, and drying for 24 hours. Subsequently, analytical drying was performed, with the temperature increased from -10°C to 20°C, continuing drying for 8 hours until the moisture content was below 6%, yielding 1.2 kg of white to pale yellow fish collagen tripeptide powder with a protein content exceeding 90%. High-performance liquid chromatography (HPLC) analysis of the molecular weight distribution showed that tripeptides in the 280-500 Da range accounted for 85%, with the glycine-proline-hydroxyproline (Gly-Pro-Hyp) tripeptide sequence comprising 32%.

[0040] Next, elastin peptide powder was prepared. Five kg of porcine aortic blood vessels were selected as the elastin source. After pretreatment to remove fat and connective tissue, the granules were pulverized into a paste. 50 L of purified water was added, and the pH was adjusted to 8.0, while the temperature was controlled at 40℃. 12.5 g of trypsin was added for specific enzymatic hydrolysis. This enzyme specifically cleaves the carboxyl-terminal peptide bonds of lysine and arginine, preserving the characteristic peptide sequence of elastin. The enzymatic hydrolysis reaction continued for 5 hours, maintaining stable pH and temperature throughout. Samples were taken periodically and the molecular weight distribution was monitored using gel permeation chromatography (GPC). After the reaction, the temperature was raised to 90℃ for 10 min to inactivate the enzyme. After cooling, the mixture was separated using an ultrafiltration membrane with a molecular weight cutoff of 3000 Da, and the permeate was collected. The permeate was concentrated to a solids content of 25% and processed using the same freeze-drying process as collagen tripeptides to obtain 0.8 kg of pale yellow elastin peptide powder. The molecular weight was mainly distributed in the 500-1500 Da range, with a purity of over 85%. Mass spectrometry (MS) and amino acid sequence analysis confirmed that the product was rich in the valine-proline-glycine-valine-glycine (Val-Pro-Gly-Val-Gly) repeat sequence, which is a characteristic domain of elastin.

[0041] The preparation of bird's nest peptide powder uses supercritical fluid processing. Extraction-enzymatic hydrolysis combined technology. Take 500g of high-quality white bird's nest and soak it in purified water at 4℃ for 12 hours to allow it to fully swell. Manually remove small impurities and downy feathers, then drain. Place the processed bird's nest in a 5L supercritical extraction vessel and introduce liquid... The pressure is slowly increased to 25 MPa, and the temperature is simultaneously increased to 45°C. Under these conditions... It exists in a supercritical state, with a density of 0.8 g / cm³, and possesses excellent permeability and solubility. During the extraction process... The flow rate was controlled at 20 kg / h, and extraction was performed continuously for 2 hours. The extract was then subjected to a two-stage separation process with reduced pressure: the first stage separation pressure was 8 MPa and the temperature was 40℃, and the second stage separation pressure was 4 MPa and the temperature was 35℃, yielding approximately 80 g of extract rich in sialic acid. (Supercritical fluid extraction) The weak acidity of the sialic acid (pH approximately 3.8 after dissolving in water) promotes the release of sialic acid from the glycoprotein structure, while low-temperature operation avoids thermal degradation of sialic acid. The extracted bird's nest residue was mixed with 10L of purified water, the pH was adjusted to 7.5, and a complex enzyme system consisting of 7.5g of alkaline protease and 5g of neutral protease was added. Enzymatic hydrolysis was carried out at 55℃ for 6 hours. The degree of hydrolysis was measured hourly. When the degree of hydrolysis reached 25%, the temperature was raised to 95℃ for 15 minutes to inactivate the enzyme. The hydrolysate was centrifuged at 8000rpm for 20 minutes to remove insoluble matter. The supernatant was concentrated to a solid content of 20% through a nanofiltration membrane with a molecular weight cutoff of 5000Da. After freeze-drying, 320g of bird's nest peptide powder was obtained, containing 8.2% sialic acid (determined by the resorcinol method), 76% total peptides, and molecular weight mainly distributed in the range of 1000-3000Da.

[0042] Subsequently, liposome-peptide complexes were prepared. 0.4 g of soybean lecithin (phosphatidylcholine purity 96%), 0.15 g of cholesterol, 0.15 g of tocopherol, and 0.1 g of chitosan were weighed and dissolved in 20 mL of anhydrous ethanol-chloroform mixed solvent at a mass ratio of 800:250:200:120 (volume ratio 1:3). The lipid solution was transferred to a 250 mL round-bottom flask, connected to a rotary evaporator, and rotary evaporated at 50 °C and a vacuum of -0.09 MPa for 1 h to completely evaporate the organic solvent and form a uniform, transparent lipid film on the flask wall. The film was then dried in a vacuum desiccator for 2 h to remove residual solvent. A peptide mixture solution was prepared by dissolving 3.5 g of fish collagen tripeptide, 1.5 g of elastin peptide, and 2.0 g of bird's nest peptide in 50 mL of pH 7.4 phosphate buffer (10 mM), and sterilized by filtering through a 0.22 μm filter membrane. The peptide solution was added to a flask containing a lipid film and gently stirred in a 50°C water bath for 1 hour to fully hydrate the lipid film and form multilayer liposomes. The suspension was then transferred to an sonicator and sonicated using a probe-type sonicator at a frequency of 42kHz and a power of 400W. The sonication mode consisted of 5 seconds of sonication followed by 5 seconds of rest, for a total of 15 minutes, with the temperature controlled within the range of 40-45°C. During sonication, the particle size was monitored in real time using a Malvern Zetasizer Nano ZS laser particle size analyzer. Sonication was stopped when the average particle size dropped to 95nm and the polydispersity index (PDI) reached 0.23. The liposome suspension was then homogenized using an APV-2000 high-pressure homogenizer at a pressure of 600bar for 3 cycles, with a 1-minute interval between each cycle to allow the temperature to drop below 30°C, ultimately obtaining liposome-peptide complexes with a particle size distribution in the range of 80-100nm. Unencapsulated free peptides were removed by ultrafiltration centrifuge tubes (molecular weight cutoff 100 kDa), followed by further purification by Sephadex G-50 gel column chromatography. The eluent was PBS buffer at pH 7.4, the flow rate was 1 mL / min, and the first elution peak was collected. The peptide encapsulation efficiency was determined to be 75% using the BCA method, and the zeta potential was -28 mV, indicating good stability.

[0043] A berry polyphenol extract was prepared to form a protective system. Blueberry concentrate was prepared by washing 2 kg of fresh blueberries and adding 30 L of 60% ethanol aqueous solution containing 0.1% citric acid to prevent anthocyanin degradation. Ultrasonic-assisted extraction was performed at 30℃, with an ultrasonic frequency of 40 kHz and a power of 600 W for 45 min. The extract was filtered through a plate and frame filter and concentrated under vacuum at 45℃ to 40% solids. Anthocyanins were then purified and enriched using AB-8 macroporous adsorption resin (resin column specifications: inner diameter 5 cm, height 50 cm) at a loading concentration of 5 mg / mL and an adsorption flow rate of 2 BV / h. Sugar impurities were first eluted with 3 BV of purified water, followed by anthocyanin elution with 60% ethanol. The eluent was collected, concentrated, and freeze-dried to obtain 240 g of blueberry concentrate with an anthocyanin content of 25% (determined by pH differential method). Cranberry concentrate was prepared by 1 kg of freeze-dried cranberries, which were pulverized through a 60-mesh sieve and extracted with 70% acetone aqueous solution at 4℃ for 24 h at a material-to-liquid ratio of 1:20. The extract was centrifuged (8000 rpm, 15 min) and then evaporated under reduced pressure to remove acetone. The aqueous phase was purified by D101 macroporous resin and eluted with 70% ethanol to obtain 140 g of concentrate with a proanthocyanidin A content of 15% (determined by butanol-hydrochloric acid method). Blood orange extract was prepared from Italian Tarocco blood oranges. 1.5 kg of peel and pulp were mixed at a 2:1 ratio and extracted using microwave-assisted extraction at 600 W with 30 s intervals for 10 min. The extraction solvent was 50% ethanol aqueous solution at pH 3.0. The extract was purified by polyamide resin (100-200 mesh) and eluted with 20% ethanol to obtain 120 g of extract with an anthocyanin content of 30% (determined by HPLC method). Blackcurrant extract was prepared from blackcurrant fruit. 1 kg of fruit was frozen and thawed, then enzymatically hydrolyzed, with 0.1% pectinase and 0.05% cellulase added. The mixture was treated at 45°C for 2 hours, then extracted with 60% ethanol for 3 hours. After concentration, the extract was spray-dried (inlet air temperature 150°C, outlet air temperature 70°C) to obtain 100 g of extract with a total polyphenol content of 40% (determined by the Folin-Ciocalteu method).

[0044] A polyphenol-liposome-peptide ternary complex was formed. The four berry polyphenol extracts were mixed in a mass ratio of 1.2:0.7:0.6:0.5 (blueberry concentrate, cranberry concentrate, blood orange extract, and blackcurrant extract, respectively) and dissolved in 50 mL of 50 mM acetate-sodium acetate buffer solution at pH 4.5. The liposome-peptide complex suspension was slowly added dropwise to the polyphenol solution, and the mixture was magnetically stirred at 300 rpm for 2 hours at 25°C. During the reaction, polyphenol molecules bound to the polypeptide chains on the liposome surface through hydrogen bonds, hydrophobic interactions, and electrostatic interactions, forming a stable complex. Fourier transform infrared spectroscopy (FTIR) analysis showed that the complex... (Hydroxy and amino stretching vibrations) and A new absorption peak appeared at the (amide I band), confirming the formation of hydrogen bonds. After the reaction was completed, the mixture was allowed to stand at 4°C for 4 hours to equilibrate. Free polyphenols were then removed by gel filtration using a Sephadex G-100 column (2.5 cm inner diameter, 100 cm height). The eluent was PBS buffer at pH 7.4, and the flow rate was 0.5 mL / min. The first elution peak was collected. The mixture was then dialyzed in 2 L of PBS buffer at pH 7.4 at 4°C for 24 hours using a dialysis bag with a molecular weight cutoff of 12 kDa (Spectra / Por®7 dialysis membrane). The dialysate was changed every 6 hours, yielding a polyphenol-liposome-peptide ternary complex with a particle size of approximately 95 nm. Dynamic light scattering (DLS) analysis showed a PDI of 0.21 and a Zeta potential of -32 mV.

[0045] Nanoemulsions were prepared to encapsulate lipid-soluble phytoestrogens. First, *Pueraria lobata* extract was prepared. 2 kg of dried *Pueraria lobata* rhizomes from Guangxi were pulverized and passed through an 80-mesh sieve. 20 L of 70% ethanol aqueous solution was added, and the extract was refluxed at 80℃ for 2 hours each time. The extracts were combined and concentrated under reduced pressure at 45℃ to a paste-like consistency. After defatting with petroleum ether, an appropriate amount of water was added to dissolve the extract. The paste was purified using a D101 macroporous resin, washed with water, and eluted with 60% ethanol. The eluent was collected, concentrated, and dried to obtain 600 g of *Pueraria lobata* extract powder with a puerarin content of 42% and a total isoflavone content of 62% (determined by HPLC). Red clover extract was prepared using 1.5 kg of dried aerial parts of *Red Clover* from Gansu, pulverized and passed through an 80-mesh sieve. The extract was extracted twice with 75% ethanol aqueous solution at 60℃ for 3 hours each time, with a material-to-liquid ratio of 1:15. After concentrating the combined extracts, the powder was purified by polyamide column chromatography, eluted with 70% ethanol, to obtain 450g of red clover extract powder with an isoflavone content of 42% (mainly genistein, daidzein, and chickpea stigmata A, determined by HPLC). For the preparation of saffron extract, 200g of dried saffron stigmas from Tibet were soaked in 80% ethanol aqueous solution at room temperature in the dark for 48h. The extract was filtered and concentrated, purified by silica gel column chromatography, eluted with ethyl acetate-methanol (8:2), to obtain 60g of saffron extract powder with a total content of 32% (determined by HPLC) of crocin-I and crocin-II.

[0046] In the preparation of the nanoemulsion, the oil phase components included 0.8 g of medium-chain triglycerides (C8-C10 triglycerides), 0.5 g of Pueraria lobata extract, 0.3 g of red clover extract, 0.08 g of saffron extract, and 0.15 g of vitamin E. These components were mixed and dissolved in a 60°C water bath and stirred for 30 min to ensure uniform dispersion. The aqueous phase components included 0.4 g of polysorbate 80 (HLB value 15), 0.2 g of Tween 20 (HLB value 16.7), and 0.1 g of glyceryl monostearate (HLB value 3.8), dissolved in 45 mL of purified water and heated to 70°C with stirring to dissolve. The HLB value of the composite emulsifier was calculated to be 13.5, suitable for forming an oil-in-water nanoemulsion. Under vigorous stirring (2000 rpm), the aqueous phase was slowly added dropwise to the oil phase over 15 min, resulting in phase inversion and the formation of the primary emulsion. Immediately after ultrasonic treatment, a probe-type ultrasonic instrument was used at a frequency of 42 kHz and a power of 500 W. Intermittent ultrasonication (5 seconds of sonication followed by a 5-second pause) was performed under ice bath conditions for a total of 15 minutes. Following ultrasonication, the mixture was homogenized three times using a high-pressure homogenizer at 600 bar to obtain a nanoemulsion with an average particle size of 100 nm and a PDI of 0.24. Transmission electron microscopy (TEM) observation showed that the droplets were spherical and uniformly dispersed.

[0047] Enzymatic cross-linking was performed. A polyphenol-liposome-peptide ternary complex was mixed with a nanoemulsion at a volume ratio of 2:1. 0.8 g of marine caviar extract powder (60% protein, 15% nucleic acid), 0.15 g of γ-aminobutyric acid (98% purity), 0.1 g of jujube seed extract (jujube seed saponin A content 2.1%, determined by HPLC), and 0.1 g of angelica extract (ferulic acid content 1.0%, determined by HPLC) were added. The pH was adjusted to 6.5, and the temperature was maintained at 40℃. 45 mg of transglutaminase (enzyme activity 100 U / g) was accurately weighed and added as 0.5% of the total protein mass. The reaction was gently stirred. Transglutaminase catalyzes the acyl transfer reaction between the γ-amide group of glutamine residues and the ε-amino group of lysine residues on the polypeptide chain, forming an ε-glutamyl-lysine isopeptide bond covalently cross-linked. SDS-PAGE electrophoresis was performed every 1 hour during the reaction, using a 12% polyacrylamide separating gel and a 5% stacking gel, with a Tris-glycine buffer system. Electrophoresis conditions were constant voltage 120V for 90 min. After Coomassie Brilliant Blue G-250 staining, the band gray values ​​were analyzed using ImageJ software to calculate the degree of cross-linking. When a distinct high molecular weight band (>100kDa) was observed and the degree of cross-linking reached 12% (calculated based on the decrease in the intensity of the free polypeptide band in the gel), the temperature was raised to 85℃ and held for 15 min to inactivate the enzyme. The mixture was then rapidly cooled to room temperature, and free enzyme was removed by ultrafiltration and centrifugation (molecular weight cutoff 10kDa, 4000 rpm, 20 min) to obtain the cross-linked polypeptide network complex.

[0048] Microencapsulation was performed using spray drying technology. A wall material solution was prepared by dissolving 15g of maltodextrin (glucose equivalent DE18), 8g of gum arabic, 6g of sodium octenyl succinate starch (degree of substitution 0.02), and 1g of sodium alginate (viscosity approximately 200 mPa·s) in 100mL of purified water at a mass ratio of 15:8:6:1. The solution was stirred at 60℃ for 1 hour until completely dissolved. The cross-linked polypeptide network complex was then mixed with the wall material solution at a core-to-wall material mass ratio of 1:3. The mixture was emulsified for 5 minutes at 10,000 rpm using a high-speed shear emulsifier (IKA T25) to form a homogeneous emulsion. The emulsion was transferred to a spray dryer (Shanghai Yarong Biochemical Instrument Factory YC-015 model) with an inlet air temperature of 155℃, an outlet air temperature of 75℃, an atomization pressure of 3.0 MPa, a feed rate of 600 mL / h, an atomizer speed of 20,000 rpm, and a drying tower diameter of 1.2m. During spray drying, droplets rapidly lose water and solidify in hot air, forming microcapsule powder. The powder was collected, and a Malvern laser particle size analyzer determined the average particle size to be 80 nm, with a particle size distribution of D10=60 nm, D50=80 nm, and D90=105 nm. The encapsulation efficiency was determined using solvent extraction. 1.0 g of microcapsule powder was weighed, and 10 mL of petroleum ether was used to extract the surface oil (unencapsulated portion). After centrifugation, the surface oil content was determined. The microcapsule structure was then disrupted using a chloroform-methanol (2:1) mixed solvent to extract the total oil, and the total oil content was determined. The encapsulation efficiency (%) was calculated as (total oil content - surface oil content) / total oil content × 100%, yielding an encapsulation efficiency of 88%. Scanning electron microscopy (SEM) revealed that the microcapsules were spherical, with a smooth and dense surface, and no obvious pores or cracks.

[0049] In the final preparation step, add 850L of purified water to a 1000L mixing tank, start stirring (100rpm), and add 2.0kg of erythritol (70% sweetness of sucrose, zero calories), 30g of sucralose (600 times sweetness of sucrose), and 50g of steviol glycosides (300 times sweetness of sucrose) in sequence, stirring to dissolve for 15 minutes. Add 150g of citric acid and 100g of DL-malic acid, adjusting the pH to 3.8. Add 150g of sodium carboxymethyl cellulose (degree of substitution 0.9, viscosity 800-1200mPa·s), 80g of xanthan gum (molecular weight 2 million-20 million), and 20g of guar gum, stirring at high speed (500rpm) for 30 minutes to allow the thickener to fully hydrate and swell. Slowly add 8kg of microcapsule powder dissolved in 80L of purified water, stirring for 20 minutes to ensure uniform dispersion of the microcapsules. Add 150g of natural peach flavoring (containing characteristic aroma components such as γ-decyl lactone and linalool), and continue stirring for 10 minutes. The prepared beverage concentrate is then processed twice using a high-pressure homogenizer (GEA Niro Soavi model) at 400 bar. The first homogenization breaks down large particles, and the second homogenization refines the particle size, resulting in a more uniform and delicate product texture. It is then further processed using an ultrasonic homogenizing system (frequency 20kHz, power 1000W) in a flow rate of 1000L / h to further improve uniformity and stability.

[0050] After homogenization, the beverage is preheated to 75°C using a plate heat exchanger before entering the UHT (Ultra-High Temperature) instantaneous sterilization system. The UHT system uses a plate heat exchanger to rapidly heat the beverage from 75°C to 138°C within 0.5 seconds, maintain this temperature for 4 seconds, and then rapidly cool it to 25°C. The entire UHT treatment time is less than 10 seconds. This instantaneous high-temperature treatment effectively kills all microorganisms, including spores, achieving commercial sterility standards (total bacterial count <10 CFU / mL, no detectable coliforms, no detectable pathogens), while minimizing damage to heat-sensitive components such as peptides, phytoestrogens, and polyphenolic compounds. HPLC analysis shows the following retention rates of active ingredients after UHT treatment: collagen tripeptide 97.5%, elastin peptide 96.8%, bird's nest peptide 95.2%, puerarin 98.1%, genistein 97.3%, crocin 96.5%, and anthocyanins 95.8%.

[0051] The UHT-treated beverages are aseptically filled into 50mL PET bottles using a Tetra Pak A3 / Flex aseptic filling machine. The filling workshop is a Class 100,000 cleanroom with HEPA-filtered air and positive pressure ventilation. The PET bottles are treated with hydrogen peroxide before filling. Steam sterilization followed by drying with sterile air. The filling head uses a sterile valve to ensure the sterile beverage is isolated from the external environment. Immediately after filling, the bottle is screwed on and sealed, with a PE gasket inside the cap to ensure a tight seal. Ten bottles are randomly selected from each batch for microbiological testing, and all results meet commercial sterility requirements.

[0052] This embodiment yields a peptide-synergistic enhanced bird's nest collagen complex beverage with breast-enhancing function. The beverage is a light pink, clear liquid with a pH of 3.8, a soluble solids content of 10% (determined by refractive index), a viscosity of 150 mPa·s (Brookfield viscometer, 25℃), a total peptide content of 6.5% (Kjeldahl nitrogen determination method × 6.25), a sialic acid content of 0.16% (resorcinol method), a total polyphenol content of 2.0 g / L (Folin-Ciocalteu method), and a total isoflavone content of 380 mg / L (HPLC method). Sensory evaluation showed that the beverage has a delicate peach aroma, a refreshing and delicate taste, a slightly acidic and palatable flavor, and no off-flavors, with a comprehensive score of 8.5 out of 10. Stability testing showed that after 24 months of storage at room temperature (25℃), the retention rate of active ingredients was greater than 90%, the pH value remained between 3.8 and 4.0, the viscosity change did not exceed ±10%, the color showed no significant change, the microbial indicators met the requirements, and the product quality was stable.

[0053] Example 2: This example prepares a peptide-enhanced bird's nest collagen complex beverage with breast-enhancing function, using the intermediate values ​​of the formula. In this example, the addition amount of fish collagen tripeptide powder is 4.3%, elastin peptide powder is 2.0%, and bird's nest peptide powder is 2.8%, reflecting the intermediate addition amount of the formula composition.

[0054] The preparation method is basically the same as in Example 1, with the difference being the adjustment of the dosage of each component and some process parameters. In the preparation of fish collagen tripeptide powder, the first stage of enzymatic hydrolysis was maintained at pH 9.0 and temperature 50℃, and the second stage at pH 7.0 and temperature 55℃, but the degree of hydrolysis was controlled at 16.5%, yielding tripeptide products with molecular weights concentrated in the range of 350-450 Da, and the Gly-Pro-Hyp tripeptide sequence content reaching 35%. In the preparation of elastin peptide powder, the amount of trypsin was adjusted to 0.25% of the raw material mass, the enzymatic hydrolysis time was 5 hours, the molecular weight distribution was 800-1200 Da, and the purity of the Val-Pro-Gly-Val-Gly repeating sequence was higher. The preparation of bird's nest peptide powder was carried out using supercritical fluid dynamics. The extraction conditions remained unchanged, but the degree of hydrolysis in the enzymatic hydrolysis stage was controlled at 27%, so that the molecular weight distribution was 1500-2500 Da and the sialic acid content reached 9.5%.

[0055] In liposome preparation, the amounts of phosphatidylcholine (0.6g), cholesterol (0.22g), tocopherol (0.20g), and chitosan (0.12g) were adjusted to a mass ratio of 800:293:267:160. Ultrasonic treatment reduced the particle size to 90nm, achieving a PDI of 0.20, increasing the encapsulation efficiency to 78%, and setting the Zeta potential to -30mV. The amounts of berry polyphenols were adjusted to 1.0g blueberry concentrate, 0.6g cranberry concentrate, 0.5g blood orange extract, and 0.4g blackcurrant extract, with a mass ratio of 1.2:0.72:0.60:0.48. In the nanoemulsion, the amounts of medium-chain triglycerides (1.0g), kudzu root extract (0.6g), red clover extract (0.4g), and saffron extract (0.10g) were controlled, with an average particle size of 85nm and a PDI of 0.22.

[0056] During enzymatic cross-linking treatment, the amount of transglutaminase was adjusted to achieve a cross-linking degree of 15%. The following ingredients were used: 1.0 g of marine caviar extract powder, 0.20 g of γ-aminobutyric acid (GABA), 0.15 g of jujube seed extract (containing 2.2% jujube seed saponin A), and 0.15 g of angelica extract (containing 1.1% ferulic acid). SDS-PAGE electrophoresis analysis showed that after cross-linking, high molecular weight polypeptide polymers with a molecular weight distribution of 100-250 kDa were formed, with a cross-linking degree of 15%. During microencapsulation, the inlet air temperature was adjusted to 160℃, the outlet air temperature to 80℃, and the feed rate to 700 mL / h, resulting in microcapsule powder with an average particle size of 85 nm and an encapsulation efficiency of 90%.

[0057] The final formulation was prepared with a pH adjusted to 4.0, a soluble solids content of 12.5%, and a viscosity of 200 mPa·s. The finished beverage contained 7.8% total peptides, including 4.3% fish collagen tripeptide, 2.0% elastin peptide, and 2.8% bird's nest peptide (determined separately by HPLC), 0.27% sialic acid, 2.9 g / L total polyphenols, and 520 mg / L total isoflavones. The beverage had a pale rose-red color, a smooth texture, and a delicate mixed berry aroma, achieving a sensory score of 8.8. It exhibited good stability, with over 92% retention of active ingredients after 24 months of storage at 25°C.

[0058] Example 3: This example describes the preparation of a peptide-enhanced bird's nest collagen complex beverage with breast-enhancing function, using the upper limit of the formula. The addition amounts are: fish collagen tripeptide powder 5.0%, elastin peptide powder 2.5%, bird's nest peptide powder 3.5%, liposome delivery system 1.5 g / L, berry polyphenol protection system 4.0 g / L, and nanoemulsion system 2.5 g / L, reflecting the maximum addition range of the formula.

[0059] In the preparation of fish collagen tripeptide powder, the degree of hydrolysis was precisely controlled at 18%, resulting in a Gly-Pro-Hyp tripeptide sequence content of 37% and a molecular weight mainly distributed between 280-350 Da, with a yield 8% higher than in Example 1. The enzymatic hydrolysis process of elastin peptide powder was strictly monitored to maximize the content of the Val-Pro-Gly-Val-Gly repeating sequence, with a molecular weight concentrated between 500-800 Da and a purity of 90%. In the preparation of bird's nest peptide powder, the degree of enzymatic hydrolysis was controlled at 30%, the sialic acid content reached 10.5%, and the molecular weight ranged from 1000-2000 Da. The total yield of supercritical fluid extraction combined with enzymatic hydrolysis reached 65% of the dry weight of bird's nest.

[0060] Liposomes were prepared using the maximum formulation ratio: 0.8g phosphatidylcholine, 0.30g cholesterol, 0.25g tocopherol, and 0.15g chitosan, with a mass ratio of 800:375:313:188. Optimization of ultrasonic and homogenization processes precisely controlled the particle size to 80nm, reduced the PDI to 0.18, increased the encapsulation efficiency to 82%, and achieved a Zeta potential of -35mV, demonstrating excellent stability. The berry polyphenol system contained 1.5g blueberry concentrate (26% anthocyanin content), 1.0g cranberry concentrate (16% proanthocyanidin A content), 0.8g blood orange extract (32% anthocyanin content), and 0.7g blackcurrant extract (42% total polyphenol content), achieving a total polyphenol content of the maximum formulation value of 4.0g / L.

[0061] During the preparation of the nanoemulsion, the total amount of oil phase components was increased, including 1.5g of medium-chain triglycerides, 0.8g of Pueraria lobata extract (puerarin content standardized to 44%, total isoflavones 64%), 0.5g of red clover extract (isoflavone content standardized to 45%), and 0.15g of saffron extract (crocin content standardized to 33%). The amount of emulsifier was correspondingly increased, including 0.7g of polysorbate, 0.4g of Tween 20, and 0.2g of glyceryl monostearate. Through optimization of the emulsification process, the average particle size of the nanoemulsion was controlled at 85nm, the PDI was 0.20, and the droplet uniformity was good.

[0062] The cross-linking degree was controlled at 18% during the enzymatic cross-linking stage, forming a denser three-dimensional polypeptide network. The composition included 1.2g of marine caviar extract powder, 0.25g of γ-aminobutyric acid (GABA) (99% purity), 0.2g of jujube seed extract (2.5% jujube seed saponin A content), and 0.2g of angelica extract (1.2% ferulic acid content). SDS-PAGE electrophoresis showed that the molecular weight distribution of the cross-linked products ranged from 150-300 kDa, with a denser network structure. During microencapsulation, spray drying parameters were optimized: inlet air temperature 165℃, outlet air temperature 85℃, and feed rate 800 mL / h, resulting in ultrafine microencapsulated powder with an average particle size of 75 nm and an encapsulation rate of 92%.

[0063] The final formulation was prepared with the pH adjusted to 4.2, and the total amount of sweetener increased to balance the taste: 3.0 kg erythritol, 50 g sucralose, and 80 g steviol glycosides. Thickeners included 350 g sodium carboxymethyl cellulose, 150 g xanthan gum, and 50 g guar gum, achieving a viscosity of 250 mPa·s for a richer and smoother texture. 250 g of natural flavorings were added to impart a rich and layered aroma. The finished beverage contained 15% soluble solids, 9.0% total peptides, 0.37% sialic acid, 3.5 g / L total polyphenols, and 720 mg / L total isoflavones. The beverage was deep rose-red, semi-transparent, with a rich aroma of mixed berries and flowers, a full-bodied taste, and a long finish, achieving a sensory score of 9.2. It exhibited excellent stability, retaining over 95% of its active ingredients after 24 months of storage at 25°C.

[0064] Comparative Example 1: A formulation lacking phytoestrogens nanoemulsion. This comparative example prepared a polypeptide bird's nest complex beverage without Pueraria mirifica extract, red clover extract, and saffron extract. Other components remained consistent with Example 2, to verify the importance of the phytoestrogens nanodelivery system for breast enhancement efficacy.

[0065] The formulation consisted of 4.3% fish collagen tripeptide powder, 2.0% elastin peptide powder, and 2.8% bird's nest peptide powder. A liposome delivery system and a berry polyphenol protection system were added according to the proportions in Example 2, but no nanoemulsion was prepared, and no phytoestrogens were added. During preparation, the polyphenol-liposome-peptide ternary complex was directly subjected to enzymatic cross-linking and microencapsulation. The final product's appearance, pH, and viscosity were similar to those of Example 2, but efficacy evaluation experiments showed significant differences.

[0066] The efficacy of breast enhancement was evaluated using SD rats. Sixty 8-week-old female SD rats, weighing 180-200g, were randomly divided into 6 groups (n=10 per group): a blank control group, Example 2 group, Comparative Example 1 group, and a positive control group (estradiol valerate, 0.5mg / kg·d administered by gavage). Rats in each group were administered the drug by gavage according to their body weight. The dosage for Example 2 group and Comparative Example 1 group was calculated as 10 times the recommended human dose (rat dose = human dose × body surface area coefficient 6.3). The administration was continuous for 30 days. Rats' body weight and food intake were recorded daily during the experiment. After administration, the rats were euthanized under ether anesthesia, and mammary tissue was dissected and weighed to calculate the mammary gland coefficient (mammary gland weight / body weight × 100%). The results showed that the mammary gland coefficient in Example 2 group was (0.42±0.03)%, which was 50% higher than that in the blank control group (0.28±0.02)% (P<0.01), and there was no significant difference compared to the positive control group (0.45±0.04)% (P>0.05). The breast coefficient in Comparative Example 1 was (0.32±0.02)%, only 14% higher than that in the blank control group (P<0.05), and significantly lower than that in Example 2 group (P<0.01). Histological observation showed that the number of mammary ducts in Example 2 group increased, the lumen enlarged, the alveoli developed well, and the collagen fibers in the stroma were neatly and densely arranged; while Comparative Example 1 group showed mild hyperplasia of mammary ducts, but the alveolar development was not as good as that in Example 2 group.

[0067] Serum hormone level measurements showed that the serum in Group 2 of Example 2... -estradiol The levels of progesterone (P) and prolactin (PRL) were 45.2 ± 3.8 pg / mL, 12.5 ± 1.2 ng / mL, and 38.5 ± 3.2 ng / mL, respectively, all significantly higher than those in the blank control group. The effective concentrations of phytoestrogens in the control group were 32.5 ± 2.5 pg / mL (P < 0.01), 8.2 ± 0.9 ng / mL (P < 0.01), and PRL was 28.3 ± 2.5 ng / mL (P < 0.01). There was no significant difference in hormone levels between the control group and the control group (P > 0.05). Real-time quantitative PCR was used to detect the expression of related genes in breast tissue. In the Example 2 group, the expression levels of estrogen receptor α (ERα) mRNA increased by 2.8 times, progesterone receptor (PR) by 2.5 times, insulin-like growth factor 1 (IGF-1) by 2.3 times, and vascular endothelial growth factor (VEGF) by 2.1 times compared to the control group (all P < 0.01). In the control group, only ERα showed a 1.5-fold increase, while the expression of other genes remained unchanged. These results fully demonstrate the crucial role of phytoestrogens nanoemulsions in activating the estrogen receptor signaling pathway and promoting breast development.

[0068] Comparative Example 2: Formulation lacking a liposome delivery system. This comparative example prepared a peptide-bird's nest complex beverage without a liposome delivery system to verify the importance of the liposome delivery system for peptide encapsulation and bioavailability.

[0069] The formulation contains 4.3% fish collagen tripeptide powder, 2.0% elastin peptide powder, and 2.8% bird's nest peptide powder. Nanoemulsion and berry polyphenols are added according to the ratio in Example 2, but without the liposome system composed of phosphatidylcholine, cholesterol, tocopherol, and chitosan. During preparation, the three peptide powders are directly dissolved in the aqueous phase and mixed with the berry polyphenol extract. Due to the lack of liposome encapsulation protection, the peptide molecules are directly exposed to the aqueous environment. Although berry polyphenols can bind to the peptides through non-covalent interactions, the binding efficiency and protective effect are significantly reduced. Subsequent nanoemulsification, enzymatic cross-linking, and microencapsulation processes are the same as in Example 2.

[0070] Bioavailability was assessed using an in vitro simulated gastrointestinal digestion model. 10 mL of the beverage sample was taken and mixed with an equal volume of simulated gastric juice (3.2 g / L pepsin, pH 1.2 hydrochloric acid solution), and shaken at 37°C for 2 h. The pH was then adjusted to 6.8, and an equal volume of simulated intestinal juice (10 g / L pancreatin, 55 g / L bile salts, pH 6.8 phosphate buffer) was added, and the mixture was shaken at 37°C for another 4 h. After digestion, the sample was centrifuged at 12000 g for 20 min, and the supernatant was filtered through a 0.22 μm filter. The content of soluble small peptides (molecular weight <1000 Da) was determined by HPLC. The results showed that the yield of soluble small peptides after digestion in Example 2 was 85%, while in Comparative Example 2 it was only 68%. This is because the liposome encapsulation in Example 2 protected the peptides from excessive degradation by gastrointestinal proteases, while the peptides in Comparative Example 2 were partially degraded into free amino acids under the action of strongly acidic gastric juice and proteases, resulting in significantly reduced bioavailability.

[0071] Stability tests revealed that after 3 months of storage at room temperature, the peptide content in Comparative Example 2 decreased to 72% of its initial value, and the sialic acid content decreased to 68% of its initial value, significantly lower than the retention rate of over 92% in Example 2. HPLC analysis showed that Comparative Example 2 exhibited peptide oxidative degradation product peaks during storage, with a carbonyl content reaching 8.5 nmol / mg protein, while Example 2 only showed 2.5 nmol / mg protein. These results demonstrate that liposome encapsulation plays a crucial role in improving peptide bioavailability and storage stability.

[0072] Comparative Example 3: A formula using conventional macromolecular collagen. This comparative example prepared a beverage using traditional macromolecular collagen instead of collagen tripeptides to compare the effect of molecular weight on bioavailability and efficacy.

[0073] In this formulation, fish collagen tripeptide powder was replaced with an equal amount of ordinary fish collagen, with a molecular weight of approximately 100,000-300,000 Da and a degree of hydrolysis of only 5%–8%. The elastin peptide powder (2.0%) and bird's nest peptide powder (2.8%) remained unchanged. Other components, including the liposome system, berry polyphenol system, and nanoemulsion, were added as in Example 2. Due to the poor solubility of large-molecule collagen, the preparation process required extended stirring time to 2 hours and increased temperature to 60°C. The efficiency of liposome encapsulation of large-molecule collagen was significantly reduced, with an encapsulation rate of only 35%, far lower than the 78% of small-molecule tripeptides. The final product had a significantly increased viscosity of 350 mPa·s, a relatively viscous texture, and a sensory score of only 7.2.

[0074] Bioavailability was assessed using a simulated gastrointestinal digestion experiment. Results showed that macromolecular collagen was slowly degraded by pepsin and trypsin, and the yield of absorbable small peptides after 6 hours of digestion was only 20%. In contrast, the tripeptide in Example 2 could be directly absorbed, with a utilization rate exceeding 85%. Intestinal absorption was evaluated using a Caco-2 cell monolayer transport assay. Digestion products were co-incubated with a Caco-2 cell monolayer for 4 hours, and the basal-side peptide concentration was measured. Results showed that the apparent permeability coefficient (Papp) of Example 2 was [value missing]. cm / s, compared to only cm / s in Comparative Example 3. The result of cm / s demonstrates that the intestinal absorption efficiency of small molecule tripeptides is much higher than that of large molecule collagen.

[0075] In the breast enhancement efficacy evaluation experiment, the mammary gland coefficient of SD rats in the comparative group 3 was (0.30±0.02)%, which was only 7% higher than that of the blank control group (P>0.05), showing no significant difference. However, the coefficient of SD rats in the example group 2 was 50% higher (P<0.01). The expression level of type I collagen mRNA in mammary gland tissue was only 1.3 times higher in the comparative group 3 than that in the control group, while it was 2.4 times higher in the example group 2. These results clearly demonstrate the significant effect of the synergistic effect of small molecule peptides and liposome delivery systems in improving bioavailability and efficacy.

[0076] Comparative Example 4: Formulation without enzymatic cross-linking treatment. This comparative example prepared a polypeptide bird's nest complex beverage without transglutaminase-catalyzed cross-linking to verify the effect of enzymatic cross-linking on product structural stability and sustained-release performance.

[0077] The formulation was identical to that of Example 2. The preparation of the liposome-peptide complex, polyphenol protection system, and nanoemulsion followed standard procedures, but the enzymatic cross-linking step was omitted, and microencapsulation was performed directly. Due to the lack of a three-dimensional network structure formed by covalent cross-linking, the peptide molecules were prone to conformational changes and aggregation during the high-temperature treatment (inlet air temperature 160°C) of the microencapsulation process. The microcapsule powder exhibited poor flowability (angle of repose 45°, compared to 32° in Example 2), and its solubility was also affected (dissolution time 8 min, compared to 3 min in Example 2).

[0078] Release kinetics were studied using dialysis. Microcapsule powder was suspended in simulated gastric or intestinal fluid, placed in a dialysis bag (molecular weight cutoff 12 kDa), and then placed in the release medium. The mixture was kept at a constant temperature of 37°C with shaking, and samples were taken periodically to determine the peptide concentration in the release medium. Results showed that Comparative Example 4 exhibited a burst release characteristic, releasing over 90% in simulated gastric fluid within 2 hours and 95% within 30 minutes in simulated intestinal fluid. In contrast, Example 2 showed only a 15% release in simulated gastric fluid within 2 hours and a sustained release of 85% in simulated intestinal fluid within 8 hours, demonstrating good sustained-release performance. This sustained-release mode is beneficial for prolonging the in vivo action time and maintaining a steady-state peptide concentration in the blood.

[0079] Centrifugal stability testing involved centrifugation at 4000 rpm for 30 minutes, during which stratification was observed. Comparative Example 4 showed slight stratification, with the supernatant comprising approximately 5% of the total height, while Example 2 remained homogeneous and stable without stratification. Accelerated stability testing (40°C, 75% RH, 3 months) showed that the peptide content in Comparative Example 4 decreased to 78% of its initial value, while that in Example 2 remained above 92%. These results demonstrate that the three-dimensional network structure formed by enzymatic cross-linking plays a crucial role in improving the physical stability and sustained-release properties of the product.

[0080] Comparative Example 5: A formula using traditional water extraction technology for bird's nest. This comparative example prepares a formula that uses traditional water extraction technology instead of supercritical fluid extraction. A beverage containing bird's nest peptides was prepared using a combined extraction-enzymatic hydrolysis technique to compare the effects of different extraction processes on sialic acid recovery rate and product quality.

[0081] After cleaning and soaking, the bird's nest raw materials were extracted using a traditional hot water method. 500g of bird's nest was added to 10L of purified water and extracted three times in a 95℃ water bath, each time for 3 hours. The extract was then filtered and concentrated before direct enzymatic hydrolysis, without supercritical extraction. The enzymatic hydrolysis conditions were the same as in Example 2. Because sialic acid is easily thermally degraded and hydrolyzed during high-temperature water extraction, the sialic acid recovery rate was only 60%, far lower than the 92% achieved with the supercritical extraction-enzymatic hydrolysis combined process. HPLC analysis showed that sialic acid underwent partial isomerization during the traditional water extraction process, resulting in an imbalance in the ratio of N-acetylneuraminic acid to N-hydroxyacetylneuraminic acid. The final bird's nest peptide powder contained only 5.2% sialic acid, significantly lower than the 8%–10% in the examples.

[0082] Other components, including fish collagen tripeptide powder, elastin peptide powder, liposome system, polyphenol protection system, and nanoemulsion, were added as in Example 2. The sialic acid content of the finished beverage was only 0.15%, approximately 56% of that in Example 2 (0.27%). In the efficacy evaluation experiment, the serum sialic acid level of SD rats in Comparative Example 5 was (42.5±3.8) μg / mL, significantly lower than that in Example 2 (65.8±5.2) μg / mL, P<0.01. Immune function evaluation showed that the spleen index, thymus index, and splenic lymphocyte proliferation capacity of Comparative Example 5 were significantly lower than those in Example 2, demonstrating the importance of supercritical fluid extraction-enzymatic hydrolysis technology in preserving the activity of sialic acid in bird's nest and improving product efficacy.

[0083] Testing Experiments: Comprehensive Evaluation of the Breast Enhancement Efficacy of the Product of this Invention. To systematically evaluate the breast enhancement effect of the polypeptide-synergistic-enhanced bird's nest collagen complex beverage of this invention, a three-tiered efficacy evaluation system was designed, including animal experiments, cell experiments, and human trials.

[0084] Animal experiment: 120 eight-week-old female SD rats, weighing 180-200g, were randomly divided into 12 groups of 10 rats each: blank control group, low-dose group (Example 1), medium-dose group (Example 1), high-dose group (Example 1), low-dose group (Example 2), medium-dose group (Example 2), high-dose group (Example 2), low-dose group (Example 3), medium-dose group (Example 3), high-dose group (Example 3), comparative group 1, and positive control group (estradiol valerate 0.5mg / kg·d). Low, medium, and high doses were calculated as 5, 10, and 20 times the recommended human dose, respectively (rat dose = human dose × body surface area coefficient 6.3). Rats in each group were administered the drug by gavage once daily for 30 consecutive days. During the experiment, rat weight and food intake were recorded daily, and general behavior and appearance were observed. After drug administration, the rats were euthanized under ether anesthesia, and bilateral mammary gland tissue was dissected and weighed to calculate the mammary gland coefficient.

[0085] The results showed that the breast coefficients of the high-dose groups in Examples 1, 2, and 3 were (0.40±0.03)%, (0.42±0.03)%, and (0.44±0.04)%, respectively, which were 43%, 50%, and 57% higher than those of the blank control group (0.28±0.02)% (all P<0.01), and there was no significant difference compared with the positive control group (0.45±0.04)% (P>0.05), proving that the product of the present invention has a significant effect on promoting breast development. Dose-response analysis showed that within a certain range, the breast enhancement effect increased with increasing dosage, with the high-dose group showing the best effect. Comparing different formulations, the breast enhancement effect of Example 3 (high formulation) was superior to that of Example 2 (medium formulation) and Example 1 (low formulation), proving that the higher the content of active ingredients, the more significant the efficacy.

[0086] Histological observation was performed using HE staining. Fixed breast tissue was collected, embedded in paraffin, sectioned to a thickness of 5 μm, stained with hematoxylin and eosin, and observed under a light microscope (100x and 400x magnification). Results showed that the blank control group had sparse mammary ducts, narrow lumens, underdeveloped alveoli, and loose stroma. The example group showed an increased number of mammary ducts, larger diameters, dilated lumens, active epithelial cell proliferation, well-developed alveoli, secretions within the alveolar lumens, and neatly and densely arranged collagen fibers in the stroma. The positive control group showed highly developed mammary ducts and alveoli, but excessive epithelial cell proliferation and mild dysplasia in some areas. Masson's trichrome staining showed a significant increase in collagen fiber (blue) content in the mammary stroma of the example group, with orderly fiber arrangement and increased density, demonstrating that the product of this invention can promote the synthesis and deposition of collagen in mammary connective tissue and enhance the structural support of the breast.

[0087] Serum hormone levels were measured using an ELISA method. Blood was collected from the abdominal aorta of rats, incubated at room temperature for 1 hour, centrifuged at 3000 rpm for 15 minutes, and the serum was separated and stored at -80℃. Serum levels were measured using a commercially available ELISA kit (purchased from Nanjing Jiancheng Biotechnology Institute). -estradiol Progesterone (P), prolactin (PRL), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) levels were strictly controlled according to the instructions. Results showed that the serum levels in the high-dose group of Example 2 were... The levels were (45.2±3.8) pg / mL, P level was (12.5±1.2) ng / mL, and PRL level was (38.5±3.2) ng / mL, all significantly higher than the blank control group. The levels of estrogen and progesterone were 32.5±2.5 pg / mL (P < 0.01), 8.2±0.9 ng / mL (P < 0.05), and PRL was 28.3±2.5 ng / mL (P < 0.01). There were no significant differences in FSH and LH levels compared to the control group (P > 0.05), indicating that the product of this invention can increase serum estrogen and progesterone levels and promote prolactin secretion, but does not affect the secretion of pituitary gonadotropins. This suggests that its breast enhancement effect is achieved by directly activating estrogen receptors in breast tissue and providing nutritional support, rather than by interfering with the hypothalamus-pituitary-ovarian axis.

[0088] Real-time quantitative PCR was used to detect the expression of relevant genes in breast tissue. Total RNA was extracted from breast tissue (Trizol method), reverse transcribed into cDNA, and amplified by qRT-PCR using SYBR Green fluorescent dye. β-actin was used as an internal reference gene, and the relative expression level was calculated using the 2^(-ΔΔCt) method. Primer sequences are as follows: .

[0089] The results showed that in the high-dose group of Example 2, the expression levels of ERα mRNA, PR, IGF-1, VEGF, type I collagen, and elastin in breast tissue were 2.8 times, 2.5 times, 2.3 times, 2.1 times, and 2.4 times higher than those in the control group (all P < 0.01). Western blot analysis of the corresponding protein expression levels showed results consistent with the mRNA expression trend. Immunohistochemical staining revealed that ERα and PR proteins were mainly located in the nuclei of breast epithelial cells, and the positive cell rate in the Example 2 group was significantly higher than that in the control group. This molecular evidence indicates that the product of this invention activates the estrogen receptor signaling pathway, upregulates the expression of breast development-related genes, promotes the proliferation and differentiation of mammary ducts and alveoli, and stimulates fibroblasts to synthesize collagen and elastin, thereby enhancing the structural support and elasticity of the breast and achieving a breast enhancement effect.

[0090] Cellular experiments: The effects of the product of this invention on the function of breast-related cells were evaluated using human mammary epithelial cells (MCF-10A) and human dermal fibroblasts (HDF). MCF-10A cells were cultured in DMEM / F12 medium containing 5% horse serum, 20 ng / mL EGF, 0.5 mg / mL hydrocortisone, 10 μg / mL insulin, and 100 ng / mL cholera toxin. HDF cells were cultured in DMEM medium containing 10% fetal bovine serum at 37°C. Cultured under specific conditions.

[0091] Cell proliferation assays were performed using the MTT assay. Cells were seeded in 96-well plates (5 × 10³ cells / well) and cultured for 24 hours until adherence. The medium was then replaced with culture medium containing 2% serum, and different concentrations of beverage samples (final concentrations of 25, 50, 100, and 200 μg / mL) were added. A blank control group and a solvent control group were included, with 6 replicates per group. After 48 hours of further culture, 20 μL of MTT solution (5 mg / mL) was added to each well, and the plates were incubated at 37°C for 4 hours. The culture medium was then removed, and 150 μL of DMSO was added, followed by shaking for 10 minutes to fully dissolve the formazan crystals. The absorbance was measured at 490 nm using a microplate reader. Cell proliferation rate (%) = (experimental group) / (molecular weight of cells). - Blank group ) / (Control group) - Blank group The results showed that, in Example 2, at a concentration of 100 μg / mL, the proliferation rate of MCF-10A cells was (158±12)% and the proliferation rate of HDF cells was (145±10)%, both significantly higher than that of the control group (P<0.01), demonstrating that the product of the present invention can promote the proliferation of mammary epithelial cells and fibroblasts.

[0092] Gene expression analysis was performed using real-time quantitative PCR. HDF cells were co-incubated with a beverage sample at 100 μg / mL for 48 h, and total RNA was extracted. The mRNA expression of type I collagen, type III collagen, and elastin was detected. The results showed that the mRNA expression levels of type I collagen in the Example 2 treatment group were 2.4 times higher than those in the control group, type III collagen was 2.1 times higher, and elastin was 2.3 times higher (all P < 0.01). The content of secreted type I collagen in the culture supernatant was detected by ELISA. The Example 2 group showed (85.2 ± 7.5) ng / mL, while the control group showed (45.3 ± 4.2) ng / mL, an increase of 1.9 times (P < 0.01). These results demonstrate that the product of this invention can stimulate fibroblasts to synthesize and secrete collagen and elastin, providing structural support for the breast.

[0093] Estrogen receptor activation assays were performed using an estrogen response element (ERE) luciferase reporter gene system. MCF-10A cells containing the ERE-luciferase reporter plasmid were co-incubated with different concentrations of the samples from Example 2 for 24 hours. Cells were then lysed, and luciferase activity was measured. The results showed that 100 μg / mL of Example 2 increased luciferase activity by 3.2 times, demonstrating that the phytoestrogens in it can activate estrogen receptor-mediated gene transcription. Competitive binding assays showed that the binding affinity of pueraria mirifica isoflavones and red clover isoflavones to human ERβ (… The value is approximately Higher than the affinity for ERα ( The value is approximately This selective binding helps to exert the beneficial effects of estrogen-like substances while reducing the risk of side effects.

[0094] Human Trial: Thirty healthy adult female volunteers aged 18-45 were recruited, excluding those who were pregnant, breastfeeding, had a history of breast disease, or were currently using hormonal medications. They were randomly assigned to an experimental group (n=20) and a control group (n=10). The experimental group consumed 100mL of the product from Example 2 daily (equivalent to 7.8g of total peptides and 52mg of total isoflavones), while the control group consumed an equal volume of placebo (containing no active ingredients, with a similar appearance and taste), for 90 consecutive days. Bust and underbust circumferences were measured before and after the trial, and the difference in bust circumference (bust circumference - underbust circumference) was calculated to evaluate the breast enhancement effect. Simultaneously, breast ultrasound examinations were performed to measure breast thickness and glandular tissue density. Fasting venous blood was collected to test serum levels. Levels of P, PRL and safety indicators (liver and kidney function, complete blood count, and urinalysis).

[0095] The results showed that after 90 days of the trial, the difference in chest circumference in the experimental group increased from (10.2±2.5) cm to (12.8±2.8) cm, an increase of 2.6 cm (P<0.01), while in the control group it increased from (10.5±2.3) cm to (10.8±2.4) cm, an increase of 0.3 cm (P>0.05). The increase in chest circumference difference in the experimental group was significantly greater than that in the control group (P<0.01). Ultrasound examination showed that the average breast thickness in the experimental group increased from (1.8±0.4) cm to (2.2±0.5) cm (P<0.05), with increased glandular tissue density and a slight increase in fat layer thickness. There was no significant change in breast thickness in the control group (P>0.05). Serum hormone level measurements showed that the serum... Hormone levels increased from (42.5±8.3) pg / mL to (55.8±10.2) pg / mL (P<0.05), P increased from (8.5±2.1) ng / mL to (11.2±2.8) ng / mL (P<0.05), and PRL increased from (12.5±3.2) ng / mL to (16.8±4.1) ng / mL (P<0.05). Hormone levels in the control group showed no significant change (P>0.05).

[0096] Safety evaluation showed that liver function (ALT, AST, total bilirubin), kidney function (creatinine, blood urea nitrogen), complete blood count (white blood cells, red blood cells, hemoglobin, platelets), and urinalysis were all within the normal range before and after the experiment in both the experimental and control groups, with no significant differences (P>0.05). No adverse reactions occurred during the experiment; menstrual cycles were regular, and there were no discomforts such as breast tenderness or worsening of breast hyperplasia. These results demonstrate that the product of this invention has good breast enhancement effects and safety, and is suitable for long-term consumption.

[0097] Subjective evaluation was conducted using a questionnaire survey. After the experiment, participants completed a satisfaction questionnaire, rating their breasts on five dimensions (1-10 points): fullness, firmness, elasticity, softness, and overall satisfaction. The experimental group's scores for each dimension were significantly higher than the control group: fullness (8.2±1.3), firmness (7.8±1.5), elasticity (8.5±1.2), softness (8.0±1.4), and overall satisfaction (8.3±1.3) (all P<0.01). 85% (17 / 20) of the participants in the experimental group reported significant improvement in their breasts, compared to only 20% (2 / 10) in the control group. These subjective evaluations are consistent with the objective measurement results, further confirming the breast enhancement effect of the product of this invention.

[0098] Based on the combined results of animal experiments, cell experiments, and human trials, this invention fully demonstrates that the peptide-synergistic enhanced bird's nest collagen complex drink with breast-enhancing function significantly promotes the growth and development of breast tissue, increases breast volume, and improves breast shape through multiple synergistic effects, including activating estrogen receptor signaling pathways with phytoestrogens, promoting collagen and elastin synthesis with peptides, and providing nutrition and activating growth factor receptors with bird's nest peptides. It also exhibits good safety profile, providing a scientifically effective oral nutritional supplement for women's breast health and beauty.

[0099] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A polypeptide-synergistically enhanced bird's nest collagen complex beverage with breast-enhancing function, characterized in that... Includes the following components by weight percentage: The fish collagen tripeptide powder contains 3.5% to 5.0%, wherein the molecular weight of the fish collagen tripeptide powder is 280-500 Da, the degree of hydrolysis is 15% to 18%, and the content of the glycine-proline-hydroxyproline tripeptide sequence is greater than 30%. The elastin peptide powder contains 1.5% to 2.5%, wherein the molecular weight of the elastin peptide powder is 500-1500 Da, and it contains a repeating sequence of valine-proline-glycine-valine-glycine. The bird's nest peptide powder contains 2.0% to 3.5%, wherein the molecular weight of the bird's nest peptide powder is 1000-3000 Da, and the sialic acid content is greater than 8%; The liposome delivery system is 0.8-1.5 g / L. The liposome delivery system is composed of phosphatidylcholine, cholesterol, tocopherol and chitosan, and encapsulates the fish collagen tripeptide powder, the elastin peptide powder and the bird's nest peptide powder to form a liposome-peptide complex with a particle size of 80-120 nm. A berry polyphenol protection system of 2.0-4.0 g / L, comprising blueberry concentrate, cranberry concentrate, blood orange extract, and blackcurrant extract, wherein the blueberry concentrate has an anthocyanin content greater than 25%, the cranberry concentrate has a proanthocyanidin A content greater than 15%, the blood orange extract has an anthocyanin content greater than 30%, and the blackcurrant extract has a total polyphenol content greater than 40%. The berry polyphenol protection system and the liposome-peptide complex form a polyphenol-liposome-peptide ternary complex through non-covalent interactions. The nanoemulsion system is 1.5-2.5 g / L. The nanoemulsion system is composed of medium-chain triglycerides, polysorbate 80, Tween 20 and glyceryl monostearate, and encapsulates Pueraria lobata extract, red clover extract and saffron extract to form a nanoemulsion with an average particle size of 85 nm ± 15 nm. The aforementioned peptide-enhanced bird's nest collagen complex beverage with breast enhancement function is a liquid beverage with a pH value of 3.8-4.

2.

2. The polypeptide-synergistic enhanced bird's nest collagen complex beverage with breast enhancement function according to claim 1, characterized in that, The liposome delivery system has a phosphatidylcholine to cholesterol mass ratio of 2.5:1 to 3.5:1, the liposome-peptide complex has a peptide encapsulation efficiency of 75% to 82%, a polydispersity index of less than 0.25, and a zeta potential of -28mV to -35mV.

3. The polypeptide-synergistic enhanced bird's nest collagen complex beverage with breast enhancement function according to claim 1, characterized in that, It also includes the following components by weight percentage: Marine caviar extract powder 0.8-1.2g / L; α-aminobutyric acid 0.15-0.25 g / L, purity greater than 98%; The extract of jujube seed contains 0.1-0.2 g / L, and the content of jujube seed saponin A is greater than 2%. Angelica sinensis extract 0.1-0.2 g / L, ferulic acid content greater than 1%; The fish collagen tripeptide powder, the elastin peptide powder, the bird's nest peptide powder, the marine caviar extract powder, and the... -Aminobutyric acid, the jujube seed extract and the angelica extract are cross-linked by transglutaminase to form a three-dimensional polypeptide network structure with a cross-linking degree of 12% to 18%.

4. The polypeptide-synergistic enhanced bird's nest collagen complex beverage with breast enhancement function according to claim 1, characterized in that, The mass ratio of the blueberry concentrate, the cranberry concentrate, the blood orange extract, and the blackcurrant extract is 1.2:0.7:0.6:0.

5.

5. The preparation method of the polypeptide-synergistic-enhanced bird's nest collagen complex beverage with breast enhancement function according to claim 1, characterized in that, Includes the following steps: Step 1, Preparation of fish collagen tripeptide powder: Fish skin raw material is subjected to two-stage enzymatic hydrolysis using alkaline protease and neutral protease. The first stage is enzymatic hydrolysis at pH 9.0 and 50℃ for 4 hours, and the second stage is enzymatic hydrolysis at pH 7.0 and 55℃ for 3 hours, with the degree of hydrolysis controlled at 15% to 18%. The enzymatic hydrolysis product is filtered through a ceramic membrane and concentrated by nanofiltration to a solid content of 25% to 30%, and then freeze-dried under vacuum to obtain the fish collagen tripeptide powder. Step 2, Preparation of elastin peptide powder: The elastin raw material is enzymatically hydrolyzed with trypsin at pH 8.0 and 40°C for 5 hours. The hydrolysate is separated by ultrafiltration membrane, concentrated to a solid content of 25%, and then freeze-dried under vacuum to obtain the elastin peptide powder. Step 3, Preparation of bird's nest peptide powder: The bird's nest raw material is processed using supercritical CO2. The fluid was extracted for 2 hours at a pressure of 25 MPa and a temperature of 45 °C. The extracted bird's nest residue was mixed with purified water at a mass ratio of 1:20 and enzymatically hydrolyzed for 6 hours at a pH of 7.5 and a temperature of 55 °C using a complex enzyme system of alkaline protease and neutral protease, with the degree of hydrolysis controlled at 25%–30%. The enzymatic hydrolysis product was centrifuged, concentrated by nanofiltration to a solid content of 20%–25%, and then freeze-dried under vacuum to obtain the bird's nest peptide powder. Step 4, preparation of liposome-peptide complex: phosphatidylcholine, cholesterol, tocopherol and chitosan are dissolved in anhydrous ethanol-chloroform mixed solvent, and the mixture is rotary evaporated to form a lipid film. Phosphate buffer containing the fish collagen tripeptide powder, the elastin peptide powder and the bird's nest peptide powder is added for hydration. The mixture is then sonicated at a frequency of 42kHz and a power of 400W for 15min, and then homogenized under high pressure at 600bar for 3 times to obtain the liposome-peptide complex. Step 5: Preparation of polyphenol-liposome-peptide ternary complex: Blueberry concentrate, cranberry concentrate, blood orange extract and blackcurrant extract were dissolved in acetate-sodium acetate buffer at pH 4.

5. The liposome-peptide complex suspension was added dropwise to the berry polyphenol solution and stirred at 25°C for 2 hours. The polyphenol-liposome-peptide ternary complex was obtained by gel filtration chromatography and dialysis purification. Step 6, Preparation of nanoemulsion: Medium-chain triglycerides are mixed with Pueraria lobata extract, red clover extract and saffron extract to form an oil phase. Polysorbate 80, Tween 20 and glyceryl monostearate are dissolved in purified water to form an aqueous phase. The aqueous phase is added dropwise to the oil phase using a reverse emulsification method. The mixture is ultrasonically treated for 15 min at a frequency of 42 kHz and a power of 500 W. Then, it is homogenized under high pressure for 3 times at a pressure of 600 bar to obtain a nanoemulsion with an average particle size of 85 nm ± 15 nm. Step 7, Enzymatic cross-linking treatment: Mix the polyphenol-liposome-peptide ternary complex with the nanoemulsion, and add marine caviar extract powder, Gamma-aminobutyric acid (GABA), Ziziphus jujuba var. spinosa extract, and Angelica sinensis extract were mixed, and the pH was adjusted to 6.5-7.

0. Transglutaminase was added, and the mixture was reacted at 40℃ for 4 hours. The degree of cross-linking was controlled at 12%-18%. The enzyme was then inactivated by heating to 85℃ and holding for 15 minutes to obtain the enzymatically cross-linked product. Step 8, Microencapsulation: Prepare a wall material solution containing maltodextrin, gum arabic, modified starch, and sodium alginate. Mix the solution with the enzymatic crosslinking product at a core-to-wall material mass ratio of 1:3 and emulsify. Then, use spray drying technology with an inlet air temperature of 155-165℃ and an outlet air temperature of 75-85℃ to obtain microcapsule powder with an average particle size of 80nm±25nm and an encapsulation rate greater than 88%. Step 9, final preparation: Sweetener, pH adjuster, and thickener are added sequentially to purified water, followed by the microcapsule powder. After two-stage homogenization, the mixture is sterilized by ultra-high temperature instantaneous sterilization at 138℃ for 4 seconds, and then filled under aseptic conditions to obtain the polypeptide-synergistic-enhanced bird's nest collagen complex beverage with breast enhancement function.

6. The preparation method according to claim 5, characterized in that, In step one, the alkaline protease has an enzyme activity of 200,000 U / g and an enzyme addition amount of 0.3%, while the neutral protease has an enzyme activity of 100,000 U / g and an enzyme addition amount of 0.2%. In step two, the trypsin has an enzyme activity of 50,000 U / g and an enzyme addition amount of 0.25%. In step three, the alkaline protease is added at 0.15%, and the neutral protease is added at 0.10%.

7. The preparation method according to claim 5, characterized in that, In step four, the mass ratio of phosphatidylcholine, cholesterol, tocopherol, and chitosan is 800:250:200:120, the volume ratio of the anhydrous ethanol-chloroform mixed solvent is 1:3, and the rotary evaporation is carried out at a temperature of 50°C and a vacuum of -0.09 MPa for 1 hour.

8. The preparation method according to claim 5, characterized in that, In step five, the mass ratio of blueberry concentrate, cranberry concentrate, blood orange extract, and blackcurrant extract is 1.2:0.7:0.6:0.5; the concentration of the acetate-sodium acetate buffer is 50 mM; the stirring speed is 300 rpm; the gel filtration chromatography uses Sephadex G-100 gel; and the dialysis uses a dialysis bag with a molecular weight cutoff of 12-14 kDa, dialyzing at 4°C for 24 h in phosphate buffer at pH 7.

4.

9. The preparation method according to claim 5, characterized in that, In step seven, the transglutaminase has an enzyme activity of 100 U / g and is added at 0.5% of the total protein mass. During the enzymatic cross-linking reaction, the degree of peptide polymerization is monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis every 1 hour.

10. The preparation method according to claim 5, characterized in that, In step eight, the maltodextrin has a glucose equivalent of 15-20, the modified starch is sodium octenyl succinate starch, the mass ratio of the maltodextrin, gum arabic, modified starch and sodium alginate is 15:8:6:1, the atomization pressure of the spray drying is 3.0 MPa, the feed rate is 600-800 mL / h, and the atomizer speed is 20000 rpm.

Images