Preparation method and application of non-denatured elastin targeting vascular elasticity and renal function

Abstract

The application discloses a preparation method and application of non-denatured elastin targeting blood vessel elasticity and kidney function, and belongs to the technical field of functional food raw material processing. Fresh bovine anterior cruciate ligament or bovine aorta preserved in a cold chain within 24 hours after slaughter is used as raw material, and the non-denatured elastin ultrafine powder embedding object is prepared through the following steps in sequence: super-low-temperature brittle pulverization, isothermal airflow winnowing classification, low-temperature weak-acid impurity-removing protein, directional defatting through lipase and low-temperature embedding. No strong acid, strong alkali, high temperature and protease hydrolysis treatment is applied in the whole process. The oral utilization problem of elastin is converted from the hydrolysis degree in the chemical scale to the particle size and antigen integrity in the physical scale. The cross-linked elastin fiber network is broken into complete microparticles with a median particle size of 0.5-5.0 microns in the brittle state with the material temperature being lower than-150 DEG C, so that the complete microparticles fall into the M cell sampling window of the intestinal tract, collagen, proteoglycan and fat shielding epitopes are removed, and low-temperature embedding is carried out to resist gastric acid.

Description

Technical Field

[0001] This invention relates to the field of functional food ingredient processing technology, specifically to a method for preparing and applying non-denatured elastin that targets vascular elasticity and kidney function. Background Technology

[0002] The elastic reserve function of the tunica media of a large artery depends on concentric elastic plates composed of elastin. Proelastin monomers self-assemble through alternating arrangements of hydrophobic and cross-linked domains, and are then catalyzed by lysyl oxidase to form desmolybdenum and isodesmolybdenum pyridinium ring crosslinks between four lysine residue side chains, thus constructing a three-dimensional network polymer capable of withstanding hundreds of millions of stretch-retraction cycles. Radiocarbon dating suggests that the biological lifespan of human elastic fibers can reach decades. After adulthood, the synthesis of elastin in the arterial wall almost ceases; therefore, its degradation is considered the irreversible structural basis for the loss of vascular elasticity.

[0003] There are two driving forces behind this structural degeneration. First, low-grade chronic metabolic inflammation triggered by a high-sugar, high-fat diet, accompanied by obesity and insulin resistance, recruits macrophages and neutrophils via the TLR4 / NF-κB pathway in the vascular wall, leading to the secretion of large amounts of matrix metalloproteinases (MMP-2, MMP-9, MMP-12) and neutrophil elastase, which directly cleave elastin. Simultaneously, late-stage glycation end products (AGEs) in a hyperglycemic state non-enzymatically modify elastin, altering its aggregation state and mechanical properties, synergistically accelerating the functional loss of elastic fibers with inflammatory activation. Second, inflammatory senescence accompanying aging occurs. Senescent vascular smooth muscle cells and endothelial cells continuously release interleukin-6 and matrix metalloproteinases through senescence-related secretory phenotypes, creating a long-term leaky microenvironment erosion. Decreased autophagy function further leads to abnormal deposition of elastin fragments on the vessel wall, which in turn further stimulates inflammation and matrix metalloproteinase secretion.

[0004] The key point is that the degradation products of elastin are not inert waste. Elastin-derived peptides, represented by VGVAPG, have clear chemotactic and pro-inflammatory activities. They can activate monocytes / macrophages via the elastin receptor complex and drive them to M1 polarization, and can also activate Th1 and Th17 effector T cells. This creates a positive feedback loop within the blood vessel wall: elastin degradation produces elastin-derived peptides, elastin-derived peptides recruit and activate immune cells, immune cells secrete more matrix metalloproteinases, and more elastin is degraded. This allows arteriosclerosis to progress in a self-sustaining manner, clinically manifested as increased pulse wave velocity, widened pulse pressure, and elevated systolic blood pressure.

[0005] The loss of vascular elasticity is not limited to the blood vessels themselves. Decreased compliance of the large arteries allows the pulsatile pressure waves generated by cardiac ejection to be directly transmitted to the low-resistance, high-perfusion microvascular bed of the kidneys, causing glomerular pulsatile damage, endothelial shearing damage, and podocyte shedding. Furthermore, renal microvascular lesions and secondary renin-angiotensin system activation, in turn, exacerbate arterial stiffness, creating a vicious cycle in the vascular-renal axis. Cohort studies have shown that arterial stiffness can independently predict the rate of renal function decline in patients with chronic kidney disease (CKD). Predialysis levels of serum MMP-2, cathepsin S, and elastin-derived peptides in CKD patients were independently correlated with aortic pulse wave velocity and its progression. Therefore, vascular elasticity and renal function are not two isolated targets in terms of pathological mechanisms, but rather two endpoints on the same elastin degradation chain.

[0006] At the intervention level, existing technologies mainly employ the pathway of hydrolyzing elastin into soluble peptides. The following four documents reflect the typical technical status of this pathway.

[0007] Chinese patent CN109602896A discloses a composition of collagen peptides and elastin peptides, its preparation method, and its application. The document discloses that elastin is prepared from bovine ligaments, bovine heart tubes, skipjack tuna bulbs, or porcine aorta. The elastin is first degreased by soaking in sodium hydroxide, then washed, minced, and the pH adjusted to 7.5–8.5. Papain and / or the complex protease Protamex are added and the mixture is enzymatically hydrolyzed at 60 °C for 6–8 h. The enzyme is then inactivated by heating to 90 °C. The mixture is then filtered through a plate and frame filter and spray-dried to obtain elastin peptides with a molecular weight of 300–2000 Da. These peptides are then combined with collagen peptides derived from cod skin for skin improvement. Its shortcomings are as follows: First, sodium hydroxide soaking is a strong alkaline treatment, while enzymatic hydrolysis at 60 ℃ and enzyme inactivation at 90 ℃ are protease hydrolysis and high-temperature treatment. The combination of these three processes completely destroys the native conformation of the desmokine and isodesmokine covalent cross-linking network and the molecular surface signal domain of mature elastin. Second, its target product is explicitly limited to soluble peptides with a molecular weight of 300-2000 Da. This molecular weight range highly overlaps with pathological elastin-derived peptides in vivo, which constitutes the introduction of pro-inflammatory mediators rather than tolerogens into the body. Third, the entire document does not address particle size control, nor does it cover intestinal M cell sampling, antigen presentation, or regulatory T cell induction. Fourth, its efficacy verification is limited to the content of skin elastin and collagen, hyaluronic acid, hydroxyproline, and MMP3, without addressing vascular elasticity or renal function.

[0008] US Patent Application No. US20090281044A1 and its family US20150004184A1 (Invention Title: Elastin digest compositions and methods utilizing the same). This document discloses: Mammalian ligaments are taken, and after artificially removing connective tissue, fat, and muscle, they are first boiled in water to remove soluble proteins, then boiled in an alkaline solution to remove resistant proteins, followed by hydrolysis with proteinase K or human elastase, purification by cellulose, and freeze-drying to obtain a mixture of elastin peptides containing GXXPG repeat sequences, used to stimulate endogenous elastin production and improve skin elasticity and wrinkles. Its shortcomings are as follows: First, the triple treatment of boiling water, boiling alkali, and protease hydrolysis constitutes more severe denaturation conditions; second, the active substance protected by its claims is a mixture of elastin peptides containing the GXXPG motif, and VGVAPG is a typical representative of the GXXPG motif, which is also the elastin-derived peptide itself described in the background section as having chemotactic and pro-inflammatory activities and capable of driving macrophage M1 polarization via the elastin receptor complex. Therefore, this scheme inevitably introduces pro-inflammatory mediators while supplementing the so-called active peptides; third, the document does not provide any particle size parameters, does not involve oral immune tolerance and regulatory T cell induction, nor does it involve gastric acid protection; fourth, its indications are limited to the skin and do not involve vascular elasticity and renal function.

[0009] The non-patent documents are: "The role of vascular elasticity and vascular endothelial function in the process of cauliflower extraction" by Shirato Eri, published in "Functional Foods and Pharmacology" Vol. 11, No. 2, 2017, pp. 95-106; and "Elastin hydrolysate derived from fish enhances proliferation of human skin fibroblasts and elastin synthesis and improves the skin conditions" by Shiratsuchi E et al., published in "Journal of the Science of Food and Agriculture" Vol. 96, No. 5, 2016, pp. 1672-1677. The former discloses that daily oral administration of 75 mg of skipjack tuna elastin peptide derived from the arterial bulb for 16 weeks can improve pulse wave velocity and blood flow-mediated vasodilation in healthy middle-aged and elderly individuals. The latter discloses that fish-derived elastin hydrolysate can promote the proliferation of human skin fibroblasts and elastin synthesis. The shortcomings of both studies are: firstly, both interventions use enzymatically hydrolyzed elastin peptides, belonging to the same technical pathway as CN109602896A and US20090281044A1, which also disrupts the cross-linking network; secondly, their observation endpoints are limited to vascular elasticity and skin, without evaluating renal function or revealing the coupling relationship between vascular improvement and renal function improvement; thirdly, their dosage is above 75 mg / day and the intervention period is as long as 16 weeks, without providing an effective regimen at lower doses; fourthly, neither study provides any technical instruction on the action of the gut mucosal immune system in its intact antigen form.

[0010] Patent application CN102154425B, entitled "Method for Producing Non-denatured Type II Collagen," is the closest existing technology to the process route of this invention. It discloses the following steps: Impurities are removed by stirring with pepsin at a mass ratio of 1:16 at pH 6.5 and pH 2.5 for 24 h at 26–28 °C. The material is then pulverized to 150–200 mesh using a cryogenic grinder. Subsequently, it is extracted with 0.5 mol / L acetic acid and 1% pepsin at 16–18 °C for 24 h. After adjusting the pH to 6.5–7.2, it is purified by salting out with 4.0 mol / L sodium chloride and washing with deionized water to obtain non-denatured type II collagen. Its shortcomings are as follows: First, its target protein is type II collagen rather than elastin, and the two are different in cross-linking chemistry, dissolution behavior and immunological properties, so its process parameters cannot be directly applied to elastin; Second, the final particle size of its cryogenic grinding is only 150-200 mesh, which is equivalent to about 75-100 μm, one to two orders of magnitude larger than the effective sampling size of intestinal M cells, and the resulting particles cannot enter the mucosal immune system via transcytosis; Third, the document uses pepsin in both the impurity removal and extraction stages, which is a protease hydrolysis treatment, contrary to the technical solution of this invention that does not apply protease hydrolysis throughout the process; Fourth, the document does not use air classification at the same temperature as the grinding, and the material is warmed up immediately after grinding and enters the room temperature wet process, nor does it use lipase-directed defatting and low-temperature embedding, so it cannot guarantee that conformational reversion will not occur during the classification process, nor can it guarantee that the antigenic epitopes will not be destroyed in the stomach.

[0011] The four documents mentioned above collectively reflect the long-held technical convention in this field: higher degree of hydrolysis and smaller molecular weight equate to better oral absorption. However, this convention is profoundly contradictory to the molecular biological characteristics of elastin. Only about one-tenth of lysine residues in mature elastin remain free; the vast majority of lysine residues participate in multivalent covalent cross-linking after modification by lysyl oxidase. It is this cross-linking network that fixes the native conformation of the molecular surface signal domain. Studies have shown that the intact conformation of proelastin exhibits low immunogenicity, promotes macrophage polarization towards the M2 type, and increases Foxp3 levels. + Regulatory T cells; however, elastin degradation products exhibit the opposite pro-inflammatory properties. This means that while hydrolysis increases solubility, it also transforms tolerogens into pro-inflammatory agents, introducing elastin-derived peptide-like fragments homologous to pathological products into the body. The positive effects on blood vessels are partially offset by the inflammatory burden introduced by the product itself, and the ability to induce antigen-specific immune tolerance is completely lost. Therefore, it has no protective effect on the kidneys, which are located at the other end of the vascular-renal axis.

[0012] If hydrolysis is abandoned in favor of physical grinding, new obstacles arise. Elastin is in a highly elastic state at room temperature, possessing extremely strong energy dissipation and elastic recovery capabilities. Most of the impact energy from conventional mechanical grinding is absorbed by elastic deformation rather than used for fracture, making it difficult to grind to the micrometer scale. Furthermore, the frictional heat generated during grinding causes irreversible collapse of the dynamic conformational assembly of hydrophobic water regions. Differential scanning calorimetry (DSC) measurements show that the glass transition temperature of dry elastin can approach 200 °C. This high thermal stability stems from the desmokinenin cross-linked structure itself, easily leading to the misconception that elastin is heat-resistant and can be processed at room temperature without hindrance. This overlooks the fact that its biological activity actually depends on the dynamic assembly state of its inherently disordered protein-like conformational assembly in hydrophobic water regions, which can collapse under the frictional heat and shear field of room-temperature grinding. This is the underlying reason why, despite employing cryogenic grinding, progress has been limited to 150–200 mesh.

[0013] In summary, the core technical problem addressed by this invention lies in how to process highly cross-linked, mature elastin, which exhibits high elasticity at room temperature, into a particulate form capable of being completely sampled and presented by the intestinal mucosal immune system, without the application of strong acids, strong alkalis, high temperatures, or protease hydrolysis, thus allowing it to enter the body as a complete antigen rather than a nutrient substrate. Furthermore, the exposed particle surface after pulverization is covered by collagen, proteoglycans, and fats, leaving the antigenic epitopes still masked; moreover, removing the covering layer makes the exposed epitopes susceptible to premature destruction by gastric acid in the stomach. Summary of the Invention

[0014] In view of the deficiencies of the prior art, the core technical problem to be solved by the present invention is: how to process highly cross-linked, mature elastin in a highly elastic state at room temperature into micron-sized particles that can be completely sampled and presented by the intestinal mucosal immune system without the application of strong acids, strong alkalis, high temperatures and protease hydrolysis, so that the elastin can enter the intestinal immune system in a form that retains its natural cross-linked conformation and complete antigenic epitopes, thereby simultaneously improving vascular elasticity and kidney function.

[0015] The technical concept of this invention lies in transforming the oral utilization problem of elastin from a long-standing chemical-scale issue—how small to hydrolyze molecules—to a physical and immunological-scale issue—how large to fragment particles and how intact to preserve epitopes. Specifically, by utilizing the transition of elastin from an elastic to a brittle state at ultra-low temperatures, the impact energy is no longer dissipated by elastic deformation but is entirely used for network breakage. This mechanically fragments the cross-linked elastic fiber network into intact particles with a median particle size of 0.5–5.0 μm without any covalent bond chemical hydrolysis. This particle size range falls precisely within the highly efficient sampling window of M cells on the surface of Pell's aggregate lymph nodes in the intestine, allowing intact elastin particles to cross the intestinal epithelial barrier via transcytosis and be absorbed by CD103 on the basal side. +Dendritic cells take up and process this protein, inducing the production of elastin-specific Foxp3 in intestinal draining lymph nodes. + Regulatory T cells. These regulatory T cells migrate to sites of inflammation in the systemic blood vessels and kidneys via a homing mechanism mediated by α4β7 and VLA-4 integrins. On the one hand, they inhibit the autoimmune response against elastin through CTLA-4, interleukin-10, and transforming growth factor-β, downregulate macrophage MMP-12 expression, and protect matrix metalloproteinase tissue inhibitor-1 from neutrophil elastase degradation, thus disrupting the positive feedback loop of elastin degradation products-matrix metalloproteinases. On the other hand, as large artery compliance recovers, they reduce the pulsatile pressure load afferent to the glomeruli and locally inhibit monocyte / macrophage infiltration and the secretion of pro-fibrotic mediators in the kidneys, achieving a dual blockade against vascular elasticity loss and renal function damage.

[0016] To solve the above-mentioned technical problems, the first aspect of the present invention provides a method for preparing non-denatured elastin that targets vascular elasticity and renal function, comprising six steps: raw material pretreatment, ultra-low temperature embrittlement and pulverization, air separation and classification by uniform airflow, low temperature weak acid removal of impurities, lipase-directed defatting and low temperature embedding, and steps S1 to S6 are performed without strong acid, strong alkali, high temperature and protease hydrolysis treatment, as defined in claim 1.

[0017] In the preferred embodiment, a pre-freezing process of -40 to -30 °C for 1 to 3 h is set before ultra-low temperature embrittlement and pulverization; the impact pulverization is carried out in 2 to 4 stages with a feed rate of 5 to 12 kg / h; the temperature of the pulverized material is not higher than -150 °C; the classifying wheel speed of the isothermal air classifier is 3500 to 6000 r / min and the air velocity is 15 to 30 m / s; the median particle size of the collected fractions is 0.8 to 2.0 μm and the D90 is not greater than 5.0 μm; the low-temperature weak acid protein removal uses an aqueous solution of acetic acid or citric acid with a concentration of 0.02 to 0.10 mol / L, a solid-liquid ratio of 1:15 to 1:30 g / mL, and is stirred at 0 to 4 °C and 150 to 300 r / min for 4 to 10 h; the directional defatting with lipase uses triglyceride lipase EC 3.1.1.3, with an enzyme dosage of 1000 to 4000 mg / mL. U / g substrate, enzymatic hydrolysis system pH 6.5–7.5, time 2–6 h; low temperature embedding, gum arabic, maltodextrin and sodium ascorbate mass ratio 5–7:2–4:0.5–1.5, wall material aqueous solution mass concentration 10%–20%, core material to wall material mass ratio 1:2–1:4, and homogenized at 5–10 °C and 20–40 MPa.

[0018] The second aspect of the present invention provides an undenatured elastin ultrafine powder embedding material prepared by the above method, wherein the median particle size of the elastin microparticles is 0.5-5.0 μm, the elastin content on a dry basis is not less than 85%, the total content of desmosin and isodesmosin is not less than 0.8%, the degree of denaturation determined by differential scanning calorimetry is not higher than 5%, and the retention rate of desmosin and isodesmosin after incubation in artificial gastric fluid at pH 1.2 at 37 °C for 2 h is not less than 90%, and the surface of the elastin microparticles is coated with a wall material layer composed of gum arabic, maltodextrin and sodium ascorbate.

[0019] The third aspect of the present invention provides an oral composition containing the above-mentioned non-denatured elastin ultrafine powder embedding material, and the use of the embedding material or the oral composition in the preparation of health foods or special dietary foods that improve vascular elasticity and renal function, wherein the health foods or special dietary foods are oral solid dosage forms with a unit dose of 30 to 120 mg based on non-denatured elastin, and the indicated method of administration is once daily for at least 12 weeks.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows: First, compared to sodium hydroxide soaking and 60°C enzymatic hydrolysis, boiling water and boiling alkali followed by protease hydrolysis, and the use of pepsin in the impurity removal and extraction stages, this invention achieves micron-level pulverization of mature elastin under conditions that completely avoid strong acids, strong alkalis, high temperatures, and protease hydrolysis. Experimental data show that the median particle size of the sample obtained using the method of this invention is 1.2 μm and the D90 is 4.3 μm. Under the same conditions, only when the pulverization is changed to room temperature mechanical pulverization, the median particle size is 42.0 μm; when the pulverization temperature is changed to -80°C, the median particle size is 18.0 μm. Neither of these can enter the M-cell sampling window. Simultaneously, the differential scanning calorimetry denaturation degree of the sample obtained by this invention is 3.2%, while that of the room temperature mechanically pulverized sample rises to 9.6%, indicating that ultra-low temperature brittle-state pulverization improves pulverization efficiency while suppressing conformational collapse caused by frictional heat. Compared to the 150-200 mesh (approximately 75-100 μm) achieved by cryogenic grinding, the median particle size of this invention is reduced by approximately two orders of magnitude.

[0021] Secondly, this invention achieves the enrichment and exposure of antigenic epitopes without damaging the cross-linked network through a combination of three processes: isothermal airflow classification, low-temperature weak acid protein removal, and lipase-directed defatting. Experimental data show that the total content of desmokine and isodesmokine in the samples of this invention is 1.05%, which decreases to 0.62% when isothermal airflow classification is omitted. Using an in vitro M-cell enrichment intestinal epithelial model, the relative transepithelial transport of the samples of this invention, calculated as 100%, still yields 91% transepithelial transport even when the low-temperature weak acid protein removal and lipase-directed defatting are omitted; however, the Foxp3 level in mouse mesenteric lymph nodes...+ Regulatory T cells account for a significant proportion of CD4+ + The proportion of T cells decreased from 4.82% to 1.51%, indicating that removing the masking layer plays a decisive role in immune recognition rather than physical transport.

[0022] Third, this invention endows microparticles with gastric acid resistance through low-temperature encapsulation. Experimental data show that after incubation at 37°C for 2 hours in simulated gastric fluid at pH 1.2, the retention rate of desmopressin and isodesmopressin in the samples of this invention was 94.6%. When the low-temperature encapsulation step was omitted, this retention rate decreased to 46.2%, and the corresponding mouse mesenteric lymph node Foxp3... + The proportion of regulatory T cells decreased from 4.82% to 1.44%.

[0023] Fourth, in a randomized, double-blind, placebo-controlled human trial, the product of this invention simultaneously produced measurable improvements in vascular elasticity and renal function. Using a daily oral administration of 60 mg of the product containing non-denatured elastin for 12 weeks as the intervention regimen, the brachial-ankle pulse wave velocity in the experimental group decreased from 1652±128 cm / s to 1479±108 cm / s, and the pulse pressure narrowed from 58.4±6.7 mmHg to 50.2±5.6 mmHg; the estimated glomerular filtration rate increased from 68.5±10.7 mL / min / 1.73 m 2 The concentration increased to 76.8 ± 11.5 mL / min / 1.73 m 2 The urine albumin / creatinine ratio decreased from 38.6±12.4 mg / g to 22.3±8.6 mg / g; serum desmokinin and isodesmokinin, elastin-derived peptides, and MMP-2 markers decreased simultaneously, and the differences in these indicators compared with the placebo control group at the same time point were all statistically significant. In contrast, no improvement in renal function indicators was observed in the control sample with equivalent doses of enzymatically hydrolyzed elastin peptides; and compared with the 75 mg / day, 16-week regimen reported in the non-patent prior art, the present invention achieved the above effects with 60 mg / day, 12 weeks.

[0024] Fifth, there is a quantitative coupling relationship between the aforementioned vascular and renal effects. Correlation analysis showed that the decrease in brachial-ankle pulse wave velocity at 12 weeks was negatively correlated with the increase in estimated glomerular filtration rate, with a correlation coefficient of -0.58. This means that the more significant the improvement in arterial stiffness, the more significant the improvement in renal function. This coupling relationship cannot be explained by the nutritional mechanism of simply supplementing amino acids or bioactive peptide substrates, but is consistent with the action pathway constructed in this invention, which involves sampling the complete antigen via M cells, inducing antigen-specific regulatory T cells, and bidirectional blocking via the vascular-renal axis. Attached Figure Description

[0025] Figure 1 This is a process flow diagram for preparing the non-denatured elastin ultrafine powder embedding material of the present invention; Figure 2 This is a schematic diagram illustrating the mechanism of action of the non-denatured elastin ultrafine powder embedding material of the present invention in targeting vascular elasticity and renal function; Figure 3 This is a comparison diagram of the particle size distribution of the samples obtained in Example 1, Comparative Example 1, and Comparative Example 2. Figure 3 In the figure, (a) is the volume differential distribution curve. Figure 3 (b) in the figure is the cumulative volume distribution curve; Figure 4 This is a comparison chart of antigenic epitope integrity and gastric acid tolerance of the samples obtained from Examples 1 to 3 and Comparative Examples 1 to 6. Figure 4 (a) in the figure represents the total content of desmokinin and isodesmokinin, and the degree of denaturation. Figure 4 (b) represents the tolerance to artificial gastric juice, the relative amount of transepithelial transport in the in vitro M cell model, and the proportion of regulatory T cells in the mouse mesenteric lymph nodes; Figure 5 The graph shows the time-series changes in vascular elasticity indices of the two groups of subjects in Example 2. Figure 5 In the figure, (a) represents the brachial-ankle pulse wave conduction velocity. Figure 5 In the diagram, (b) represents the pulse pressure difference; Figure 6 The graph shows a comparison of renal function indicators before and after intervention in the two groups of subjects in Example 2. Figure 7 The diagram shows the changes in serum elastin metabolic markers and the correlation between blood vessels and kidneys in the two groups of subjects in Example 2. Figure 7 In the table, (a) represents the serum marker level relative to baseline. Figure 7 (b) in the figure is a scatter plot showing the correlation between the change in brachial-ankle pulse wave conduction velocity and the estimated change in glomerular filtration rate. Detailed Implementation

[0026] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise stated, all reagents used are commercially available analytical grade or food grade, and the raw materials, bovine nuchal ligament and bovine aorta, are obtained from slaughter by-products of healthy beef cattle that have passed inspection and quarantine.

[0027] Undenatured elastin refers to mature elastin that has not undergone strong acid, strong alkali, high temperature, or protease hydrolysis during its preparation process, and whose native conformation of the desmoplastic and isodesmoplastic cross-linked structure and molecular surface signal domains is preserved. This invention uses the degree of denaturation determined by differential scanning calorimetry as a quantitative criterion for the degree of undenaturedness, and the total content of desmoplastic and isodesmoplastic components as a proxy indicator for the integrity of the cross-linked network.

[0028] Material temperature: refers to the measured temperature of the material being crushed within the crushing chamber. It is continuously monitored at the discharge port of the crushing chamber using an embedded platinum resistance temperature sensor, and is distinct from the liquid nitrogen gas phase temperature or the chamber wall temperature. The statement that the material temperature is below -150℃ means that this measured value remains below -150℃ throughout the entire crushing process.

[0029] Particle size determination: The sample was dispersed in anhydrous ethanol and ultrasonically dispersed for 3 min. The particle size was then determined by volume distribution using a laser particle size analyzer. The median particle size and D90 were reported. The median particle size refers to the particle size corresponding to 50% of the cumulative volume distribution.

[0030] Determination of total content of desmolide and isodesmolide: The sample was hydrolyzed with 6 mol / L hydrochloric acid at 110 °C for 24 h, and then quantified by liquid chromatography-tandem mass spectrometry. The mass fraction was calculated with the dry basis mass of the sample as the denominator. It should be noted that this hydrolysis step is only a necessary sample pretreatment for analysis and is not part of the preparation process of this invention.

[0031] Determination of denaturation: Take 5 mg of dried sample and place it in an aluminum crucible. Use a differential scanning calorimeter to heat the sample from 25 °C to 250 °C at a rate of 10 °C / min under a nitrogen atmosphere. Record the enthalpy change of the characteristic endothermic peak of elastin. Based on the enthalpy change of the standard non-denatured elastin reference, the degree of denaturation is calculated as (1 - enthalpy change of sample / enthalpy change of reference) × 100%.

[0032] Artificial gastric fluid tolerance test: Prepare artificial gastric fluid with pH 1.2 according to the Pharmacopoeia of the People's Republic of China. Take a sample equivalent to 60 mg of non-denatured elastin and add it to 50 mL of artificial gastric fluid. Incubate at 37 ℃ and 100 r / min for 2 h with shaking. Centrifuge and collect the solid phase. Measure the content of desmosin and isodesmosin. The ratio of the content to the content before incubation is the retention rate.

[0033] Determination of relative transepithelial transport in an in vitro M-cell model: A model of M-cell enrichment intestinal epithelium was established by co-culturing Caco-2 cells and Raji B cells and inducing them with RANKL. The sample was added to the apical side and incubated for 4 h. The amount of elastin microparticles on the basal side was measured. The transport amount of the sample in Example 1 was taken as 100%, and the remaining samples were converted proportionally.

[0034] Determination of the proportion of regulatory T cells in mouse mesenteric lymph nodes: Six- to eight-week-old male C57BL / 6 mice were administered a daily dose equivalent to 3 mg / kg body weight of non-denatured elastin via gavage for 14 consecutive days. Mesenteric lymph nodes were isolated 24 hours after the last administration, and Foxp3 levels were measured by flow cytometry. + Regulatory T cells account for a significant proportion of CD4+ + The percentage of T cells.

[0035] The range of values ​​for each process parameter in this invention is determined through end-value tests, and the technical basis for this is as follows.

[0036] Regarding the freshness of raw materials, materials stored within 24 hours of slaughter and at a cold chain below 4°C throughout the entire process are selected. After 24 hours, endogenous proteases and microbial proteases begin to act on the connecting regions of the elastic fiber network. The measured total content of desmokine and isodesmokine decreased from 1.05% to below 0.79%, falling below the quality control limit of this invention; the same phenomenon occurs when the cold chain temperature is above 4°C. Preferably, feed is given within 12 hours of slaughter, at which time the total content of desmokine and isodesmokine can be stably maintained above 1.00%.

[0037] Regarding the side length of the tissue blocks, a length of 3–5 mm is preferred. If the length is less than 3 mm, the mechanical shearing and heating during the cutting process itself damages the surface elastic fibers, and the tissue blocks are easily lost during rinsing. If the length is greater than 5 mm, the liquid nitrogen heat transfer path is too long, and the material temperature in the core of the block is still above -150 °C when it enters the grinding chamber, resulting in incomplete embrittlement and an increased proportion of coarse powder after grinding. A side length of 4 mm is preferred.

[0038] Regarding pre-freezing conditions, -40 to -30 °C for 1 to 3 hours was selected. Above -30 °C, larger ice crystals formed within the tissue, and the mechanical action of ice crystal growth tore the elastic fiber network and decreased the total content of desmolide and isodesmolide. Pre-freezing below -40 °C was harmless, but it increased liquid nitrogen consumption and did not further enhance subsequent embrittlement. If the freezing time was less than 1 hour, the core of the block was not fully frozen; if it was more than 3 hours, there was no further benefit. Pre-freezing at -35 °C for 2 hours was preferred.

[0039] Regarding the material temperature for pulverization, it is limited to below -150 °C. The transition of elastin from an elastic to a brittle state occurs in the extremely low temperature range. Actual measurements show that at a material temperature of -80 °C, the median particle size can only reach 18.0 μm, and at -120 °C, the median particle size is 7.6 μm, still not falling within the 0.5–5.0 μm window. Below -150 °C, the median particle size drops below 5.0 μm and can be further narrowed through fractionation. Further lowering the material temperature to -190 °C does not significantly decrease the median particle size, but liquid nitrogen consumption increases dramatically. The preferred material temperature is -175 °C.

[0040] Regarding the number of impact crushing stages and the feed rate, 2–4 stages and 5–12 kg / h were selected. Below 2 stages, the energy input per stage was too high, causing localized instantaneous temperature rise and increasing the denaturation degree from 3.2% to 6.9%. Above 4 stages, the crushing efficiency did not improve further, but the liquid nitrogen load on the equipment increased significantly. Below 5 kg / h, the number of impacts per unit material was too high, resulting in over-crushing and producing a large amount of debris with a particle size below 0.3 μm. Above 12 kg / h, the material residence time in the crushing chamber was insufficient, the proportion of coarse powder increased, and the temperature of the discharged material rose back above -150 ℃. A 3-stage crushing process with a feed rate of 8 kg / h was preferred.

[0041] The median particle size is limited to 0.5–5.0 μm. Below 0.5 μm, particles tend to be non-specifically taken up by intestinal epithelial cells or retained by the mucus layer, resulting in a decreased proportion of samples taken by M cells. Above 5.0 μm, particles exceed the effective size range for M cell transcytosis, and the measured relative transepithelial transport decreases from 100% to below 24%. The preferred median particle size is 0.8–2.0 μm, within which the relative transepithelial transport can be maintained above 86%, with an optimal median particle size of 1.2 μm.

[0042] Regarding the classifier rotation speed and airflow velocity, a range of 3500–6000 r / min and 15–30 m / s were selected. When the rotation speed is below 3500 r / min or the airflow velocity is below 15 m / s, the cut particle size is too large, and the D90 of the collected fraction exceeds 5.0 μm. When the rotation speed is above 6000 r / min or the airflow velocity is above 30 m / s, the fine powder loss rate increases, and the target fraction yield decreases from 68% to below 41%. A rotation speed of 4500 r / min and an airflow velocity of 22 m / s are preferred.

[0043] Regarding the pH and concentration of the weakly acidic system, a pH range of 2.5–4.0 and a concentration of 0.02–0.10 mol / L were selected. Above pH 4.0, collagen and proteoglycans did not dissolve completely, with the measured residual collagen mass fraction exceeding 6%, and antigenic epitopes were still partially masked. Below pH 2.5, elastin peptide bonds began to partially degrade, the total content of desmosin and isodesmosin decreased, and the degree of denaturation increased. Below a concentration of 0.02 mol / L, the buffering capacity was insufficient, and the system pH rose rapidly after the addition of materials. Above 0.10 mol / L, the amount of water required to wash to neutrality increased exponentially. A 0.05 mol / L acetic acid aqueous solution was preferred, at which point the system pH was 3.2.

[0044] Regarding the temperature, time, solid-liquid ratio, and rotation speed for protein removal, 0–4 ℃, 4–10 h, 1:15–1:30 g / mL, and 150–300 r / min were selected. Above 4 ℃, the acidic conditions and temperature synergistically increased the degree of denaturation; below 0 ℃, the system froze and could not be stirred. Below 4 h, dissolution was incomplete; above 10 h, residual collagen no longer decreased, while the total content of desmolide and isodesmolide began to decrease slowly. Below a solid-liquid ratio of 1:15, the system was too viscous, limiting mass transfer; above 1:30, solvent consumption and subsequent centrifugation load increased significantly. Below 150 r / min, the microparticles precipitated and agglomerated; above 300 r / min, the shear field caused secondary agglomeration of the particles. The preferred settings were 2 ℃, 6 h, 1:20 g / mL, and 200 r / min.

[0045] Regarding the dosage, temperature, pH, and time for lipase, the optimal values ​​were 1000–4000 U / g substrate, 5–15 °C, pH 6.5–7.5, and 2–6 h. When the dosage was below 1000 U / g, the residual fat mass fraction was higher than 1.8%, and the microparticle surface remained covered by a lipid film. Above 4000 U / g, the defatting rate no longer improved, but costs increased. Below 5 °C, lipase activity was too low; above 15 °C, although lipase activity was higher, measurable changes began to appear in the dynamic conformation of the hydrophobic water regions on the elastin surface, with the denaturation degree increasing from 3.2% to 5.4%. Enzyme activity decreased when the pH deviated from the 6.5–7.5 range. Defatting was incomplete below 2 h, and there was no further gain above 6 h. The preferred values ​​were 2000 U / g, 10 °C, pH 7.0, and 4 h.

[0046] Regarding the temperature, wall material ratio, wall material concentration, core-to-wall ratio, and homogenization pressure for the embedding process, the following parameters were selected: 10℃, 5–7:2–4:0.5–1.5, 10%–20%, 1:2–1:4, and 20–40 MPa. Above 10℃, the surface conformation of the particles in the wall material loosens during film formation, reducing the tolerance to artificial gastric fluid. A gum arabic ratio below 5% results in insufficient film continuity, while above 7% leads to excessive wall material viscosity, hindering homogenization. A maltodextrin ratio below 2% increases film brittleness, while above 4% causes premature swelling in gastric fluid. A sodium ascorbate ratio below 0.5% provides insufficient antioxidant protection, while above 1.5% increases system acidity, which is detrimental to film formation. A wall material concentration below 10% results in insufficient film thickness and a 2-hour retention rate of less than 80% in artificial gastric fluid; above 20% results in excessive system viscosity and decreased embedding uniformity. When the core-to-wall ratio is higher than 1:2, the wall material usage is insufficient; when it is lower than 1:4, the elastin content per unit mass of product drops below 85%. When the homogenization pressure is lower than 20 MPa, emulsification is incomplete; when it is higher than 40 MPa, shear heat makes it difficult to maintain the material temperature below 10 °C. The preferred wall material mass ratio is 6:3:1, wall material concentration is 15%, core-to-wall ratio is 1:2.5, homogenization pressure is 30 MPa, and encapsulation temperature is 8 °C.

[0047] Regarding the unit dose, 30–120 mg (based on non-denatured elastin) was selected. Below 30 mg, the amount of antigen sampled by M cells and entering the intestinal immune system was insufficient to stably induce antigen-specific regulatory T cells; above 120 mg, the improvement in vascular and renal parameters no longer increased with increasing dose, and the incidence of gastrointestinal discomfort increased. 60 mg was preferred.

[0048] Example 1 like Figure 1 As shown, this embodiment is implemented sequentially according to steps S1 to S6, and the specific operations are as follows.

[0049] S1. Raw material pretreatment. For example... Figure 1 As shown in step S1, 10.0 kg of fresh bovine neck ligament, which was stored in a cold chain at 2 ℃ for 10 h after slaughter, was taken. The attached fat and fascia were removed, and the tissue was cut into tissue blocks with a side length of about 4 mm. The tissue was washed three times with a 0.9% sodium chloride solution at 2 ℃ at a solid-liquid ratio of 1:5 to remove blood stains. The tissue was drained at 4 ℃ for 30 min to obtain 9.1 kg of pretreated tissue.

[0050] S2, ultra-low temperature embrittlement and pulverization. For example... Figure 1As shown in step S2, the pretreated tissue is spread flat in a stainless steel pan and pre-frozen in a -35 ℃ quick-freezing chamber for 2 h; then it is put into a liquid nitrogen immersion low-temperature impact mill, so that liquid nitrogen directly contacts the material. After the temperature sensor reading stabilizes at -175 ℃, the pulverization begins; the mill chamber is protected by nitrogen, the feeding rate is 8 kg / h, and the impact pulverization is carried out in 3 stages. The actual measured temperature of the material at the discharge port is -158 ℃, and coarse elastin powder is obtained.

[0051] S3, air separation and classification of the same temperature airflow. For example... Figure 1 As shown in step S3, the coarse powder, without leaving the low-temperature system, directly enters the air classifier within the same liquid nitrogen atmosphere via a pipeline. The classifier wheel speed is 4500 r / min, and the airflow velocity is 22 m / s; the density is greater than 2.0 g / cm³. 3 Sand and heavy metal particles were discharged from the coarse powder outlet, while fat and cell membrane fragments were discharged from the fine powder outlet. The intermediate fraction was collected, yielding 2.86 kg of elastin ultrafine powder, with a yield of 31.4%. The median particle size was determined to be 1.2 μm and the D90 was 4.3 μm by laser particle size analyzer.

[0052] S4. Low-temperature, weakly acidic treatment to remove impurities from proteins. For example... Figure 1 As shown in step S4, elastin ultrafine powder was added to a 0.05 mol / L acetic acid aqueous solution (measured pH 3.2) at a solid-liquid ratio of 1:20 g / mL, and stirred at 2 °C and 200 r / min for 6 h. During this time, collagen and proteoglycans gradually dissolved, while the highly cross-linked elastin remained insoluble. Subsequently, the mixture was centrifuged at 4 °C and 8000 × g for 15 min, the supernatant was discarded, and the supernatant was washed with pure water at 2 °C until the pH of the supernatant reached 6.5–7.0, yielding impurity-free elastin ultrafine powder. The residual collagen mass fraction was determined to have decreased from 18.6% before treatment to 2.4%.

[0053] S5, lipase-directed delipidemia. For example... Figure 1 As shown in step S5, the purified elastin ultrafine powder was dispersed in 0.05 mol / L phosphate buffer at pH 7.0, and triglyceride lipase (EC 3.1.1.3, enzyme activity 10000 U / g) was added until the enzyme concentration reached 2000 U / g substrate. Enzymatic hydrolysis was carried out at 10 ℃ and 150 r / min for 4 h. After centrifugation, the powder was washed three times with pure water at 2 ℃ to remove free fatty acids and residual enzymes. After pre-freezing at -80 ℃, it was freeze-dried, with the material temperature not exceeding 10 ℃ throughout the process, yielding 2.31 kg of undenatured elastin ultrafine powder. The residual fat content was determined to have decreased from 4.7% before treatment to 0.9%.

[0054] S6, low-temperature embedding. For example... Figure 1As shown in step S6, gum arabic, maltodextrin, and sodium ascorbate were weighed in a mass ratio of 6:3:1 and prepared into a 15% (w / w) aqueous solution of wall material with pure water at 8 ℃. The non-denatured elastin ultrafine powder was dispersed in the wall material solution in a core material to wall material mass ratio of 1:2.5. The solution was homogenized twice under high pressure of 30 MPa at 8 ℃, followed by spray freeze drying. The material temperature did not exceed 10 ℃ throughout the process, resulting in 8.02 kg of non-denatured elastin ultrafine powder encapsulation.

[0055] like Figure 1 The bottom box shows the finished product and quality control. The finished product was tested and found to have: a median elastin particle size of 1.2 μm, an elastin content of 88.6% on a dry basis, a total desmokine and isodesmokine content of 1.05%, a differential scanning calorimetry (DSC) thermal denaturation rate of 3.2%, a desmokine and isodesmokine retention rate of 94.6% in artificial gastric fluid after 2 hours, a residual collagen mass fraction of 2.4%, a residual fat mass fraction of 0.9%, and lead, arsenic, mercury, and cadmium content all below the corresponding national food safety standard limits. Figure 1 As indicated by the dashed box on the left, no strong acid, strong alkali, high temperature, or protease hydrolysis treatment was applied throughout steps S1 to S6.

[0056] Example 2 The difference between this embodiment and Embodiment 1 is that the raw material is changed to fresh bovine aorta, and the process parameters are taken from other values ​​within the range. The rest of the operation is the same as in Embodiment 1.

[0057] In S1, 10.0 kg of fresh bovine aorta, preserved for 18 hours post-slaughter at 4°C, was harvested. The exocarp and intima were removed, leaving the media rich in elastic lamellae. The aorta was then cut into tissue blocks approximately 3 mm in length. In S2, pre-freezing was performed at -40°C for 3 hours, with the material temperature at -155°C. The material was pulverized in two stages using impact pulverization at a feed rate of 12 kg / h. In S3, the classifier rotated at 3500 r / min, the airflow velocity was 15 m / s, and the median particle size of the collected fractions was 2.6 μm with a D90 of 4.8 μm. In S4, the weak acid system was replaced with a 0.02 mol / L citric acid aqueous solution (measured pH 3.8), with a solid-liquid ratio of 1:30 g / mL. The mixture was stirred at 4°C and 150 r / min for 10 hours. In S5, 4000 U / g of enzyme was added, and the mixture was enzymatically hydrolyzed at 15°C and pH 6.5 for 2 hours. In S6, the mass ratio of gum arabic, maltodextrin, and sodium ascorbate is 5:4:1.5, the wall material concentration is 10%, the core-to-wall ratio is 1:2, and it is homogenized at 10 °C and 20 MPa.

[0058] The obtained non-denatured elastin ultrafine powder embeddings were tested and found to have the following characteristics: median particle size of elastin microparticles 2.6 μm, elastin content 86.9% on a dry basis, total content of desmosin and isodesmosin 0.96%, denaturation degree 3.8%, and retention rate of artificial gastric fluid 93.1% after 2 hours.

[0059] Example 3 This embodiment uses a boundary scheme with parameter values ​​close to the extreme values ​​of the range. The difference from Embodiment 1 is as follows: In S3, the classifier rotation speed is 6000 r / min and the airflow velocity is 30 m / s. The fraction with a median particle size of 4.6 μm and a D90 of 5.0 μm is collected by adjusting the secondary classification cutting point; In S4, the pH of the weak acid system is 2.5, the stirring temperature is 0 ℃, the time is 4 h, and the solid-liquid ratio is 1:15 g / mL; In S5, the enzyme dosage is 1000 U / g substrate, the temperature is 5 ℃, and the time is 6 h; In S6, the mass ratio of gum arabic, maltodextrin, and sodium ascorbate is 7:2:0.5, the wall material concentration is 20%, the core-to-wall ratio is 1:4, the homogenization pressure is 40 MPa, and the remaining operations are the same as in Embodiment 1.

[0060] The obtained non-denatured elastin ultrafine powder embeddings were tested and found to have the following characteristics: median elastin particle size of 4.6 μm, elastin content of 85.3% on a dry basis, total content of desmokine and isodesmokine of 0.87%, denaturation degree of 4.6%, and retention rate of artificial gastric fluid of 91.2% after 2 hours. All indicators met the quality control requirements of this invention, but all were lower than those in Example 1, indicating that taking the median value within the specified range is more advantageous.

[0061] Comparative Example 1: Room temperature mechanical grinding. The liquid nitrogen cooling and brittle state control in step S2 were omitted. Instead, grinding was performed at 25 ℃ using an impact mill of the same model in an air atmosphere. Grinding continued until the discharge temperature reached 48 ℃, at which point further fine grinding was no longer possible. The remaining operations were the same as in Example 1. The resulting sample had a median particle size of 42.0 μm, a total content of desmolide and isodesmolide of 0.98%, a denaturation degree of 9.6%, and a 2-hour retention rate of 88.5% in artificial gastric fluid.

[0062] Comparative Example 2: Low-temperature pulverization of materials with insufficient temperature. The material temperature in step S2 was changed from -175 ℃ to -80 ℃, and the remaining operations were the same as in Example 1. The median particle size of the obtained sample was 18.0 μm, the total content of desmosin and isodesmosin was 0.94%, the degree of denaturation was 6.8%, and the retention rate of artificial gastric fluid after 2 h was 90.1%.

[0063] Comparative Example 3: Air separation and grading using the same temperature airflow were omitted. Step S3 was cancelled, and instead, the coarse elastin powder was restored to 25 °C and then sieved at room temperature using a standard test sieve. The undersize material was collected. The remaining operations were the same as in Example 1. The median particle size of the obtained sample was 3.1 μm. However, due to the failure to separate the non-elastin components and the warming of the material during sieving, the total content of desmokine and isodesmokine was only 0.62%, which is lower than the quality control limit of this invention. The denaturation degree was 7.4%, and the retention rate of artificial gastric fluid after 2 hours was 89.0%.

[0064] Comparative Example 4: Low-temperature weak acid removal of impurities and lipase-directed defatting were omitted. The wet processing in steps S4 and S5 was cancelled; the elastin ultrafine powder obtained in step S3 was directly freeze-dried and then proceeded to step S6; the remaining operations were the same as in Example 1. The resulting sample had a median particle size of 1.3 μm, a total content of desmokine and isodesmokine of 1.02%, a denaturation degree of 3.4%, a 2-hour retention rate of 93.8% in artificial gastric fluid, a residual collagen mass fraction of 18.6%, and a residual fat mass fraction of 4.7%.

[0065] Comparative Example 5: Low-temperature embedding was omitted. Step S6 was cancelled, and the non-denatured elastin ultrafine powder obtained in step S5 was used directly as the finished product; the remaining operations were the same as in Example 1. The median particle size of the obtained sample was 1.2 μm, the total content of desmosin and isodesmosin was 1.04%, the degree of denaturation was 3.3%, and the retention rate of artificial gastric fluid after 2 h was only 46.2%.

[0066] Comparative Example 6: Enzymatic hydrolysis of elastin peptides. Prepared according to the route disclosed in CN109602896A, using fresh bovine ligaments (same as in Example 1), the peptides were first soaked in a 2% sodium hydroxide solution at room temperature for 4 hours to remove fat. After washing and crushing, the pH was adjusted to 8.0, and papain was added for enzymatic hydrolysis at 60 °C for 6 hours. The enzyme was then inactivated at 90 °C for 10 minutes. After plate and frame filtration, the filtrate was spray-dried to obtain enzymatically hydrolyzed elastin peptides with a degree of hydrolysis of 23.5% and a molecular weight concentrated in the range of 300–2000 Da. The obtained sample was a soluble powder with a total content of 0.11% desmosin and isodesmosin. The cross-linked structure was basically destroyed, and the retention rate of desmosin and isodesmosin in artificial gastric juice after 2 hours was 8.4%.

[0067] Example 1: Evaluation of Particle Size Distribution and Antigenic Epitope Integrity Samples obtained from Examples 1 to 3 and Comparative Examples 1 to 6 were taken respectively, and particle size distribution, total content of desmosin and isodesmosin, degree of denaturation, tolerance to artificial gastric juice, relative amount of transepithelial transport in in vitro M cell model and proportion of regulatory T cells in mouse mesenteric lymph nodes were determined according to the aforementioned detection methods.

[0068] like Figure 3 The image shows a comparison of the particle size distribution of the samples obtained in Example 1 with those in Comparative Examples 1 and 2. Figure 3 As shown in (a), the main peak of the volume differential distribution curve of the sample in Example 1 falls entirely within the M-cell sampling window of 0.5–5.0 μm, while the main peaks of Comparative Example 2 (material pulverization at -80 °C) and Comparative Example 1 (mechanical pulverization at room temperature) are located near 18.0 μm and 42.0 μm, respectively, both completely outside the window; Figure 3As shown in (b), the median particle sizes corresponding to the 50% cumulative volume distribution curves for the three materials are 1.2 μm, 18.0 μm, and 42.0 μm, respectively. These results indicate that only when the material temperature is below -150 ℃ and the elastin enters a brittle state can the impact energy no longer be dissipated by elastic deformation and be effectively used for the mechanical fracture of the cross-linked network; when the material temperature is insufficient, even extending the pulverization time cannot push the particle size into the target window.

[0069] like Figure 4 The image shows the comparison results of antigenic epitope integrity and gastric acid tolerance for each sample. Figure 4 As shown in (a), the total content of desmodium and isodesmodium in Examples 1 to 3 was 1.05%, 0.96%, and 0.87%, respectively, all higher than the quality control limit of 0.8%, and the denaturation degree was 3.2%, 3.8%, and 4.6%, respectively. The denaturation degree of Comparative Example 1 increased to 9.6% due to frictional heating during room temperature pulverization. The total content of desmodium and isodesmodium in Comparative Example 3 dropped to 0.62% due to the omission of isothermal air separation. The total content of desmodium and isodesmodium in the enzymatically hydrolyzed elastin peptide of Comparative Example 6 was only 0.11% and the denaturation degree was close to 100% because the cross-linking structure was basically destroyed.

[0070] like Figure 4 As shown in (b), the retention rates of desmopressin and isodesmopressin in the sample of Example 1 after 2 h in artificial gastric fluid were 94.6%. The relative transepithelial transport in the in vitro M-cell model was calculated as 100%, and the Foxp3 level in mouse mesenteric lymph nodes was... + Regulatory T cells account for a significant proportion of CD4+ + The proportion of T cells was 4.82%; the corresponding values ​​for Examples 2 and 3 were 93.1%, 86%, and 4.15%, and 91.2%, 64%, and 3.06%, respectively. Although the retention rates of Comparative Examples 1 and 2 were acceptable, the relative transepithelial transport was only 11% and 24%, respectively, due to the particle size being much larger than the sampling window, and the proportion of regulatory T cells decreased to 1.28% and 1.62%, respectively.

[0071] It is noteworthy that Comparative Examples 4 and 5 exhibit two different failure modes. Comparative Example 4 omitted the low-temperature weak acid removal of impurities and the lipase-directed defatting. Its particle size, total content of desmoplastic and isodesmoplastic, degree of denaturation, and artificial gastric fluid retention rate were all close to those of Example 1, and the relative transepithelial transport also reached 91%, indicating that the physical transport of microparticles was basically unaffected. However, its regulatory T cell percentage dropped sharply from 4.82% to 1.51%. This result shows that although microparticles covered by residual collagen, proteoglycans, and fats can still pass through the intestinal epithelium, they cannot be effectively recognized and presented by the basal dendritic cells. The removal of impurities and defatting processes address the epitope accessibility rather than the particle transportability problem, and the two are not interchangeable. Comparative Example 5, which omitted low-temperature embedding, saw its artificial gastric fluid retention rate plummet to 46.2% after 2 hours, its relative transepithelial transport volume drop to 19%, and its regulatory T cell proportion decrease to 1.44%. This indicates that even if particle size and epitope accessibility meet the standards, if the particles cannot pass completely through the stomach, all the efforts of the preceding steps will be in vain.

[0072] The enzymatically hydrolyzed elastin peptides of Comparative Example 6, prepared according to the CN109602896A protocol, showed the lowest levels in all the aforementioned immunological indicators, with a regulatory T cell percentage of only 0.96%, comparable to the blank control. This result is also applicable to the mixture of elastin peptides containing the GXXPG motif obtained by hydrolysis with boiling water, boiling alkali, and proteinase K disclosed in US20090281044A1. This result directly confirms the technical concept of this invention: the oral value of elastin does not lie in its nutritional properties as an amino acid or bioactive peptide substrate, but rather in the natural conformational epitopes fixed by the cross-linked network; once hydrolyzed, it transforms from a tolerogen into a pro-inflammatory fragment homologous to pathological products in vivo, losing its ability to induce antigen-specific immune tolerance.

[0073] Example 2: Randomized, double-blind, placebo-controlled human trial 1. Trial Design. A single-center, prospective, randomized, double-blind, placebo-controlled parallel design was adopted. The entire process followed the principles of the Declaration of Helsinki and the requirements of Good Clinical Practice for drug clinical trials. All subjects signed written informed consent forms before enrollment.

[0074] 2. Subjects. Inclusion criteria were: age 50–70 years, gender not limited; brachial-ankle pulse wave velocity not less than 1400 cm / s; estimated glomerular filtration rate of 45–89 mL / min / 1.73 m 2 The creatinine-cystatin C combined formula for CKD-EPI was used for calculation; informed consent was obtained voluntarily. Exclusion criteria were: estimated glomerular filtration rate less than 45 mL / min / 1.73 m³ / min. 2Or have already received dialysis treatment; experienced an acute cardiovascular or cerebrovascular event within the past 3 months; have malignant hypertension; have nephrotic syndrome or active glomerulonephritis; have taken elastin or collagen peptide supplements within the past 3 months; have severe liver dysfunction or malignant tumors; or have a known allergy to the test ingredient.

[0075] 3. Grouping and Intervention. 120 subjects were randomly assigned in a 1:1 ratio to the experimental group and the control group using a computer-generated random sequence, with 60 subjects in each group. The experimental group received a daily oral solid dosage form prepared according to claim 9 from the non-denatured elastin ultrafine powder embedding obtained in Example 1, with a unit dose of 60 mg based on non-denatured elastin. The control group received a daily oral placebo of 60 mg of maltodextrin, which had an appearance, color, taste, and packaging completely identical to the original. Both groups took the medication with warm water for 12 consecutive weeks, maintaining their original dietary habits and basic medication regimens. Subjects, follow-up researchers, and laboratory personnel were all kept blinded.

[0076] 4. Observation indicators. The primary endpoint was brachial-ankle pulse wave velocity, measured at weeks 0, 6, and 12; secondary vascular indicators were pulse pressure, systolic blood pressure, and diastolic blood pressure; serum elastin metabolism markers were total desmokinin and isodesmokinin, elastin-derived peptides, and MMP-2; renal function indicators were estimated glomerular filtration rate, serum creatinine, blood urea nitrogen, cystatin C, and urine albumin / creatinine ratio, measured at weeks 0 and 12.

[0077] 5. Statistical methods. Quantitative data are expressed as mean ± standard deviation. Paired-samples t-tests were used for within-group comparisons, and analysis of covariance with baseline correction was used for between-group comparisons. Repeated measures ANOVA was used for comparisons at multiple time points. Pearson correlation was used for correlation analysis. A p-value < 0.05 was considered statistically significant.

[0078] 6. Results. A total of 113 patients completed all follow-up visits, including 57 in the experimental group and 56 in the control group. Seven patients dropped out due to loss to follow-up or personal reasons. No withdrawals were due to adverse events. There were no statistically significant differences between the two groups in terms of age, sex composition, body mass index, baseline brachial-ankle pulse wave velocity, pulse pressure, estimated glomerular filtration rate, serum creatinine, and urine albumin / creatinine ratio, making them comparable.

[0079] like Figure 5 The figure shows the time-series changes in vascular elasticity parameters for two groups. Figure 5As shown in (a), the brachial-ankle pulse wave velocity in the experimental group decreased gradually from 1652±128 cm / s at week 0, to 1568±116 cm / s at week 6, and to 1479±108 cm / s at week 12, a decrease of 173±42 cm / s from baseline at week 12. In the control group, the velocity decreased only slightly from 1641±135 cm / s to 1617±126 cm / s during the same period, a decrease of 24±31 cm / s. The difference between the two groups at week 12 was 138 cm / s, with a p-value < 0.01. Figure 5 As shown in (b), the pulse pressure in the experimental group narrowed from 58.4±6.7 mmHg to 50.2±5.6 mmHg, a reduction of 8.2±2.9 mmHg, while the control group only narrowed by 0.8±2.1 mmHg. The difference between the groups was P<0.01. Furthermore, the systolic blood pressure in the experimental group decreased from 141.8±9.6 mmHg to 131.2±8.1 mmHg, while the changes in diastolic blood pressure at each time point were small in both groups, and there were no statistically significant differences within or between groups. This is consistent with the hemodynamic characteristics of improved arterial elasticity primarily manifested as narrowing of pulse pressure.

[0080] like Figure 6 The figure shows the comparison results of renal function indicators before and after the intervention in the two groups. The estimated glomerular filtration rate in the experimental group increased from 68.5 ± 10.7 mL / min / 1.73 m... 2 The concentration increased to 76.8 ± 11.5 mL / min / 1.73 m 2 The increase was 8.3 ± 3.6 mL / min / 1.73 m. 2 Meanwhile, the control group's concentration decreased from 67.9±11.2 mL / min / 1.73 m during the same period. 2 The differences between groups were statistically significant (P<0.01). In the experimental group, serum creatinine decreased from 90.4±13.6 μmol / L to 80.1±11.8 μmol / L, blood urea nitrogen decreased from 6.9±1.4 mmol / L to 5.8±1.2 mmol / L, cystatin C decreased from 1.15±0.19 mg / L to 1.01±0.16 mg / L, and the urine albumin / creatinine ratio decreased from 38.6±12.4 mg / g to 22.3±8.6 mg / g. All five indicators showed statistically significant differences compared to week 0 of this group and the control group at the same time point. Figure 6 The values ​​are marked with asterisks and hash symbols respectively. In the control group, none of the renal function indicators showed significant changes compared to baseline at 12 weeks. The consistent and mutually corroborating changes in the five indicators suggest that the improvement in renal function in the experimental group exhibited cross-indicator consistency, rather than isolated fluctuations in a single indicator.

[0081] like Figure 7 The image shows the results of changes in serum elastin metabolic markers and the correlation analysis of blood vessel-renal coupling. Figure 7As shown in (a), the serum levels of desmokinin and isodesmokinin in the experimental group decreased from 0.42±0.09 μg / L to 0.31±0.07 μg / L, a decrease of 26.2%; elastin-derived peptide decreased from 68.5±14.2 ng / mL to 47.6±10.5 ng / mL, a decrease of 30.5%; and MMP-2 decreased from 218.6±38.4 ng / mL to 172.3±30.7 ng / mL, a decrease of 21.2%. The differences in all three markers compared with those in the experimental group at week 0 and the control group at the same time point were statistically significant, while the three indicators in the control group showed no significant changes at the same time point. Desmolysin, isodesmolysin, and elastin-derived peptides are cross-linking markers and degradation products reflecting the degree of elastin degradation in vivo, respectively. MMP-2 is a key protease involved in elastin degradation. The simultaneous decrease of these three constitutes a chain of evidence that mutually corroborates each other at three levels: degradation enzyme, degradation product, and cross-linking marker. This suggests that the intervention does indeed play a role in inhibiting the matrix metalloproteinase-mediated elastin degradation pathway.

[0082] like Figure 7 As shown in (b), at 12 weeks, the changes in brachial-ankle pulse wave velocity in the experimental group were significantly negatively correlated with the changes in estimated glomerular filtration rate (GFR), with a correlation coefficient of -0.58 (P < 0.01, n = 57). The scatter plots were distributed along a negative slope regression line, meaning that the more significant the decrease in brachial-ankle pulse wave velocity, the more significant the increase in estimated GFR. This result provides direct in vivo support for the vascular-renal axis mechanism, indicating that the present invention does not produce two independent effects on the blood vessels and the kidneys, but rather alleviates the pulsatile pressure load afferent to the glomeruli by restoring the common upstream link of aortic compliance.

[0083] 7. Parallel comparison with the enzymatic hydrolysis of elastin peptides described in CN109602896A. A third group of 30 subjects received 60 mg daily of an oral solid dosage form prepared from the enzymatic hydrolysis of elastin peptides obtained in Comparative Example 6, with the remaining regimens the same as above. At 12 weeks, the brachial-ankle pulse wave velocity decreased by 62±36 cm / s in this group, which was better than the placebo group but significantly less than the experimental group; the estimated change in glomerular filtration rate was 0.4±2.9 mL / min / 1.73 m 2The urine albumin / creatinine ratio changed by -1.2±6.8 mg / g, with no statistically significant difference compared to the placebo group. Serum elastin-derived peptide levels increased instead of decreasing, from 67.9±14.6 ng / mL to 74.3±15.2 ng / mL. These results indicate that an equal dose of enzymatically hydrolyzed elastin peptides only produces limited vascular effects and cannot improve renal function. Furthermore, the hydrolyzed peptides themselves introduce elastin-derived peptide-like fragments into the body, thus confirming the technical judgment of this invention regarding the conversion of tolerogens into pro-inflammatory agents through hydrolysis. This also explains why CN109602896A, US20090281044A1, and non-patent prior art documents failed to extend to the renal endpoint.

[0084] Example 3: Safety Evaluation During the intervention, two cases of mild gastrointestinal discomfort were reported in the experimental group (incidence rate 3.3%), and one case of mild gastrointestinal discomfort was reported in the control group (incidence rate 1.7%). All symptoms were transient and resolved spontaneously without specific treatment, and did not lead to participant withdrawal. There was no statistically significant difference in the incidence of adverse events between the two groups. No clinically significant abnormalities were observed in blood routine tests and liver function indicators (alanine aminotransferase, aspartate aminotransferase, etc.) before and after the intervention in either group, and no serious adverse events occurred throughout the intervention. Furthermore, the encapsulated material obtained in Example 1 was tested according to national food safety standards, and the contents of lead, arsenic, mercury, and cadmium were all below the corresponding limits. The total bacterial count, coliform bacteria, and mold and yeast counts all met the relevant regulations for solid beverages. These results indicate that the product of this invention containing 60 mg of non-denatured elastin orally daily for 12 weeks demonstrates good safety and tolerability.

[0085] like Figure 2 As shown, this illustrates the mechanism of action of the non-denatured elastin ultrafine powder embedding material of the present invention in targeting vascular elasticity and renal function. Figure 2 As shown in the five steps from top to bottom, the product of this invention completes five steps sequentially after oral administration: After the microparticles with a wall material coating enter the stomach, the coating layer resists the acidic corrosion of gastric acid. In addition, the highly cross-linked covalent network of mature elastin itself has significant resistance to pepsin and trypsin, thus preventing premature destruction of the antigenic epitopes; after entering the intestine, the microparticles with a median particle size of 0.5–5.0 μm fall precisely into the M-cell sampling window covering the surface of Pell's aggregate lymph nodes and isolated lymphoid follicles, and cross the intestinal epithelial barrier via transcytosis to reach the basal side; the CD103 on the basal side... + Dendritic cells take up and process intact elastin antigens, which are then induced to produce elastin-specific Foxp3 in intestinal draining lymph nodes via a transforming growth factor-β and retinoic acid-dependent pathway. + Regulatory T cells; these regulatory T cells migrate to the inflammatory microenvironment of the systemic blood vessels and kidneys via inflammatory homing characteristics mediated by α4β7 and VLA-4 integrins.

[0086] like Figure 2 As shown in the vascular pathway box on the left, at the vascular level, homing regulatory T cells work together to protect the elastin elastic fiber network of the tunica media through four pathways. First, they precisely inhibit the autoimmune response against elastin through CTLA-4, interleukin-10, and transforming growth factor-β, disrupting the positive feedback loop of elastin degradation producing elastin-derived peptides, elastin-derived peptides recruiting immune cells, and immune cells intensifying degradation. Second, the interleukin-10 secreted by T cells downregulates the expression of MMP-12 on macrophages, and the inhibition of Th1 and Th17 responses reduces the stimulation of macrophages by interferon-γ and interleukin-17, inhibiting the excessive secretion of MMP-12 at its source. Third, they protect the integrity and activity of matrix metalloproteinase tissue inhibitor-1 (MMI) by inhibiting neutrophil activation, restoring the enzyme-inhibition balance between MMP-12 and its tissue inhibitor. Fourth, they reduce local reactive oxygen species levels, preventing oxidative modification that alters the conformation of elastin, making it more easily recognized by MMI, thereby maintaining the physical resistance of the natural cross-linked conformation. The simultaneous decrease in serum desmokine and isodesmokine, elastin-derived peptide and MMP-2 markers in Example 2 is circulatory evidence of the combined action of the above four pathways.

[0087] like Figure 2 As shown in the right-hand kidney pathway box, at the kidney level, the protective effect comes from both the improvement of the systemic vascular environment and the local immune and anti-inflammatory effects on the kidney. With the restoration of aortic compliance, the reflected wave generated by cardiac ejection weakens, and the pulse pressure and shear stress transmitted to the afferent arterioles of the kidney decrease accordingly. This alleviates glomerular hypertension and hyperfiltration, thereby preventing glomerular capillary endothelial damage, podocyte shedding, and secondary glomerulosclerosis caused by hypertension. Simultaneously, regulatory T cells migrating to the kidney release interleukin-10 and transforming growth factor-β, directly inhibiting monocyte / macrophage infiltration and inflammatory factor secretion in the kidney tissue. This reduces the damage to the tubular basement membrane and extracellular matrix metabolic imbalance caused by the overexpression of MMP-2, MMP-9, and MMP-12 induced by transforming growth factor-β, protecting the peritubular capillary network and delaying the progression of renal interstitial fibrosis.

[0088] like Figure 2 As shown by the bidirectional arrows between the two pathways and the dashed line between the two endpoint boxes, the vascular pathway and the renal pathway are not independent of each other, but are coupled via the vascular-renal axis. The -0.58 correlation coefficient between the decrease in brachial-ankle pulse wave velocity and the estimated increase in glomerular filtration rate in Example 2 is a quantitative representation of this coupling relationship at the individual level: restoring vascular elasticity is a key intermediate step in improving renal function through oral administration of non-denatured elastin. Figure 2As shown in the lower middle section, the vascular endpoint and the renal endpoint boxes indicate that the two pathways eventually converge into measurable vascular endpoints and renal endpoints, respectively.

[0089] In summary, the technical effect of this invention stems from the inseparable causal chain between each step. Cryogenic embrittlement addresses the fundamental problem of how to break down the cross-linked network to the M-cell sampling window without hydrolysis; this is the cause. However, after fragmentation, new problems inevitably arise, such as impurities mixing with the target particles, and the material becoming unusable once it warms up. This leads to isothermal airflow classification. The surface of the classified particles is inevitably covered by collagen, proteoglycans, and fats, leading to low-temperature weak acid protein removal and lipase-directed degreasing. Furthermore, the exposed epitopes after decontamination inevitably face premature destruction by gastric acid, leading to low-temperature encapsulation. These four technical methods are progressive and mutually causal; the absence of any link leads to overall failure, a point fully verified by the five different failure modes presented in Comparative Examples 1 to 5.

[0090] The above description is merely a preferred embodiment of the present invention and does not limit the scope of patent protection of the present invention. Any equivalent structural modifications made based on the description and drawings of the present invention, or direct or indirect applications in other related technical fields, are similarly included within the scope of patent protection of the present invention.

Claims

1. A method for preparing non-denatured elastin targeting vascular elasticity and renal function, characterized in that, Includes the following steps: S1. Raw material pretreatment: Take fresh bovine nuchal ligament or bovine aorta that has been stored in a cold chain below 4 ℃ within 24 hours after slaughter, remove attached fat and fascia, cut into tissue blocks with a side length of 3-5 mm, rinse with 0.9% sodium chloride solution and drain to obtain pretreated tissue. S2. Low-temperature embrittlement and pulverization: The pretreated tissue is placed in a liquid nitrogen atmosphere and cooled to a temperature below -150°C, causing it to change from an elastic state to a brittle state. Then, it is subjected to impact pulverization under nitrogen protection to obtain coarse elastin powder. S3. Air classification at the same temperature: The coarse elastin powder is classified by air classification in the same low-temperature atmosphere as in step S2, and the fractions with a median particle size of 0.5 to 5.0 μm are collected to obtain elastin ultrafine powder. S4. Low-temperature weak acid removal of impurities from elastin: The elastin ultrafine powder is placed in a weak acid system with a temperature not higher than 4 ℃ and a pH of 2.5 to 4.0 and stirred to dissolve collagen and proteoglycan while keeping elastin insoluble. After solid-liquid separation, it is washed until neutral to obtain impurity-free elastin ultrafine powder. S5. Lipase-directed degreasing: Lipase is added to the impurity-removed elastin ultrafine powder, and enzymatic degreasing is performed at 5-15 ℃. After washing, the powder is dried at a temperature not higher than 10 ℃ to obtain non-denatured elastin ultrafine powder. S6. Low-temperature encapsulation: Using gum arabic, maltodextrin and sodium ascorbate as wall materials, the non-denatured elastin ultrafine powder is encapsulated at a material temperature not exceeding 10 ℃ to obtain non-denatured elastin ultrafine powder encapsulation material. In particular, steps S1 to S6 do not involve strong acid, strong alkali, high temperature, or protease hydrolysis.

2. The preparation method according to claim 1, characterized in that, In step S2, the pretreated tissue is first pre-frozen at -40 to -30 ℃ for 1 to 3 h, and then placed in a liquid nitrogen atmosphere for cooling; the impact crushing is carried out in 2 to 4 stages, with a feeding rate of 5 to 12 kg / h, and the temperature of the crushed material is not higher than -150 ℃.

3. The preparation method according to claim 1, characterized in that, In step S3, the classifying wheel rotation speed of the air classifier is 3500–6000 r / min, and the airflow velocity is 15–30 m / s; the median particle size of the collected fractions is 0.8–2.0 μm and the D90 is not greater than 5.0 μm; the density discharged from the coarse powder outlet during the classification process is greater than 2.0 g / cm³. 3 Inorganic impurities, such as fats and cell membrane fragments, are discharged from the fine powder outlet.

4. The preparation method according to claim 1, characterized in that, In step S4, the weak acid system is an aqueous solution of acetic acid or citric acid with a concentration of 0.02–0.10 mol / L, a solid-liquid ratio of 1:15–1:30 g / mL, a stirring temperature of 0–4 ℃, a stirring time of 4–10 h, and a stirring speed of 150–300 r / min.

5. The preparation method according to claim 1, characterized in that, In step S5, the lipase is triglyceride lipase EC 3.1.1.3, the amount of enzyme added is 1000-4000 U / g substrate, the pH of the enzymatic hydrolysis system is 6.5-7.5, and the enzymatic hydrolysis time is 2-6 h; the drying is freeze drying.

6. The preparation method according to claim 1, characterized in that, In step S6, the mass ratio of gum arabic, maltodextrin and sodium ascorbate is 5-7:2-4:0.5-1.5, the mass concentration of the wall material aqueous solution is 10%-20%, the mass ratio of core material to wall material is 1:2-1:4, and the material is homogenized at 20-40 MPa at 5-10 °C before embedding.

7. The preparation method according to any one of claims 1 to 6, characterized in that, The non-denatured elastin ultrafine powder embedding material has an elastin content of not less than 85% on a dry basis, a total content of desmosin and isodesmosin of not less than 0.8%, a denaturation degree determined by differential scanning calorimetry of not more than 5%, and a retention rate of desmosin and isodesmosin of not less than 90% after incubation at 37 °C for 2 h in artificial gastric fluid at pH 1.

2.

8. A non-denatured elastin ultrafine powder embedding material, characterized in that, The non-denatured elastin ultrafine powder embedding material is prepared by any one of claims 1 to 7; the median particle size of the elastin microparticles is 0.5 to 5.0 μm, the total content of desmodium and isodesmodium on a dry basis is not less than 0.8%, and the surface of the elastin microparticles is coated with a wall material layer composed of gum arabic, maltodextrin and sodium ascorbate.

9. An oral composition, characterized in that, The oral composition comprises the non-denatured elastin ultrafine powder encapsulation as described in claim 8 and food- or pharmaceutically acceptable excipients; the unit dose of the oral composition, based on non-denatured elastin, is 30–120 mg; the excipients are selected from at least one of microcrystalline cellulose, magnesium stearate, silicon dioxide, and maltodextrin.

10. The use of the non-denatured elastin ultrafine powder embedding material of claim 8 or the oral composition of claim 9 in the preparation of health foods or special dietary foods that improve vascular elasticity and renal function, characterized in that, The health food or special dietary food is an oral solid dosage form with a unit dose of 30-120 mg based on non-denatured elastin, and its indicated method of administration is once daily for no less than 12 weeks.

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