Natural collagen fiber and preparation method and application thereof

Abstract

This application relates to a natural collagen fiber, its preparation method, and its application. The preparation method of the natural collagen fiber includes: sequentially mixing and reacting dermal tissue particles derived from mammals with a first mixing reagent and a second mixing reagent to obtain impurity-free collagen particles; the first mixing reagent, by weight, includes water, a strong base-weak acid salt at 1%~3% by weight, and an inorganic base reagent at 0.15%~0.8% by weight; the second mixing reagent, by weight, includes water and a monohydric alcohol at 40%~50% by weight; and the impurity-free collagen particles are pulverized to obtain natural collagen fibers. This preparation method is simple, efficient, and has a high extraction rate. The obtained natural collagen fibers have a superhelical quaternary structure formed by the aggregation of natural triple helical structures, exhibiting alternating light and dark stripes; and the type I and type III collagen in the fibers are well preserved, with high protein purity.

Description

Technical Field

[0001] This application relates to the field of natural collagen fiber technology, and in particular to a natural collagen fiber, its preparation method and application. Background Technology

[0002] Collagen is a major component of the extracellular matrix, accounting for more than one-third of mammalian proteins. Types I and III collagen, the most abundant types, generally exist in the body as supramolecular aggregates of fibers. It is a key substance supporting tissue morphology and maintaining physiological function. Due to its excellent biocompatibility, bioactivity, and degradation properties, collagen-based medical materials have been widely used in various clinical fields such as wound repair, hemostasis, bone regeneration, soft tissue filling, and implantation, possessing irreplaceable application value. Natural collagen fibers are fibrous aggregates formed by the orderly aggregation of collagen molecules (approximately 300 nm in length) in a 1 / 4 staggered (axially offset by approximately 67 nm) manner, with sizes reaching the micrometer, millimeter, and even centimeter scales. This unique assembly method endows natural collagen fibers with a dense triple-helix molecular structure and a hierarchically interlaced supramolecular structure, forming multi-level fibrous structures from the nanometer to the micrometer and millimeter scales. They can even aggregate into macroscopically visible, longer, and thicker fiber bundles, giving them hydrophilic but water-insoluble properties, as well as some resistance to acids and alkalis. Compared to soluble collagen, natural collagen fibers also have the characteristics of strong plasticity and excellent molding performance in subsequent processing, making them an ideal substrate for preparing high-performance medical materials.

[0003] However, natural collagen fibers are distributed in the body in a network-like interwoven form, with various impurities such as fats, residual cells, glycoproteins, and interfibrous proteins (globulins, mucins, etc.) widely embedded in the interfibrillary spaces. Therefore, how to efficiently remove these impurities while maximally preserving the natural supramolecular structure of type I and type III collagen components and collagen fibers is one of the core technical challenges in collagen fiber extraction. In existing technologies, collagen extraction and preparation mainly use soluble collagen such as acid-soluble, alkali-soluble, and salt-soluble collagen as the main products. These methods require strong acid, strong alkali, or high salt conditions to destroy the natural aggregation structure of collagen, which not only leads to the disintegration of the supramolecular structure of collagen fibers and the loss of the unique advantages of natural collagen fibers, but also suffers from problems such as the loss of type I and type III collagen components and low extraction rates, making it difficult to meet the requirements for the preparation of high-performance medical collagen raw materials. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this application provides a natural collagen fiber, its preparation method, and its application, which effectively preserves the natural structure of collagen fibers and better retains type I and type III collagen components, while improving protein purity and fiber extraction rate.

[0005] In a first aspect, embodiments of this application provide a method for preparing natural collagen fibers, comprising the following steps: S1: Dermal tissue particles derived from mammals are sequentially mixed with a first mixing reagent and a second mixing reagent to obtain impurity-free collagen particles; wherein, based on the total mass of the first mixing reagent, the first mixing reagent includes water, a strong base-weak acid salt at a mass percentage of 1% to 3%, and an inorganic base reagent at a mass percentage of 0.15% to 0.8%; based on the total mass of the second mixing reagent, the second mixing reagent includes water and a monohydric alcohol at a mass percentage of 40% to 50%. S2: Crush the impurity-removed collagen particles to obtain natural collagen fiber impurity-removed collagen particles.

[0006] In the above technical solution, this application sequentially treats dermal tissue particles with a first mixed reagent and a second mixed reagent. The first mixed reagent causes the dermal tissue particles to swell, destroying impurities between collagen fibers; the second mixed reagent causes the dermal tissue particles to shrink, dissolving impurities. The resulting impurity-free collagen particles are compact, plump, and have an intact fiber structure. Further pulverization yields natural collagen fibers. This preparation method is simple, efficient, and has a high extraction rate. The prepared natural collagen fibers possess a superhelical quaternary structure formed by the aggregation of natural triple helical structures, exhibiting alternating light and dark stripes. Furthermore, type I and type III collagen in the fibers are well preserved, and the protein purity is high.

[0007] In the first mixed reagent, the strong base-weak acid salt is weakly alkaline. Combined with a small amount of inorganic base reagent, it forms a buffered weak base system that causes gentle swelling of collagen fibers in animal tissues, effectively removing soluble impurities such as fats, interstitial proteins, glycoproteins, and cells. Furthermore, by controlling the low concentrations of the strong base-weak acid salt and inorganic base reagent, the natural triple helix structure of collagen can be avoided, thus preserving type I and type III collagen components well.

[0008] In the second mixed reagent, the higher concentration of monohydric alcohol provides an antibacterial environment and causes dermal tissue particles to shrink and collagen fibers to dehydrate, removing impurities dissolved during the swelling process without damaging the collagen fiber structure. Furthermore, the rapidly shrinking and densifying of the swollen collagen fibers forms compact, plump, impurity-free collagen particles, effectively preventing fiber entanglement and aggregation during subsequent pulverization, thus improving the dispersibility of natural collagen fibers in subsequent processing.

[0009] In some embodiments, the strong base-weak acid salt includes at least one of sodium bicarbonate, sodium carbonate, or sodium acetate; the inorganic base reagent includes at least one of sodium hydroxide, potassium hydroxide, or calcium hydroxide; and the monohydric alcohol includes at least one of ethanol, n-propanol, isopropanol, or isobutanol.

[0010] In some embodiments, the first mixed reagent further includes an oxidant at a mass percentage of 0.5% to 1.0%. Further, the oxidant includes at least one of hydrogen peroxide or sodium hypochlorite.

[0011] In the above technical solution, an appropriate amount of oxidant is added to the first mixed reagent. The oxidant exhibits enhanced oxidizing properties under alkaline conditions, which on the one hand plays a bactericidal role, ensuring the level of microbial load and endotoxin, and more importantly, it plays a role in dissociating and dispersing collagen bundles while stabilizing the collagen fiber structure, which is conducive to further improving the dispersibility and stability of natural collagen fibers.

[0012] In some embodiments, the first mixed reagent further includes a surfactant at a mass percentage of 0.01% to 0.05%. Further, the surfactant includes at least one of anionic or cationic surfactants.

[0013] In the above technical solution, the addition of a small amount of surfactant is beneficial for the removal of immune substances such as cell debris and nucleic acids, thereby reducing immunogenicity.

[0014] In some embodiments, the second mixed reagent further includes 0.5% to 1.0% of a metal ion chelating agent. Further, the metal ion chelating agent includes at least one of disodium ethylenediaminetetraacetate or tetrasodium ethylenediaminetetraacetate.

[0015] In the above technical solution, adding a small amount of metal ion chelating agent can complex metal ions to remove metal impurities and the dissolution of other impurities, thereby further improving the purity of natural collagen fibers.

[0016] In some embodiments, the second mixed reagent further includes: 0.1% to 0.5% of an astringent; the astringent includes organic acid compounds and / or polyphenolic compounds. Further, the astringent includes at least one of tannic acid, catechins, or tea polyphenols.

[0017] In the above technical solution, polyphenolic compounds can form stable cross-links with collagen fibers, and organic acid compounds can form ionic bonds with the amino groups of collagen fibers, thereby reducing the electrostatic repulsion between fiber molecules, promoting fiber shrinkage, making the fibers dense and compact, which is beneficial to reducing fiber aggregation and entanglement during subsequent processing, thereby further improving the dispersibility of natural collagen fibers.

[0018] In some embodiments, in step S1, the reaction time of the dermal tissue particles and the first mixed reagent is 8h~12h, the temperature is 25℃~35℃, and the mass ratio of the dermal tissue particles to the first mixed reagent is 1:(2~5).

[0019] In the above technical solution, adjusting the reaction time, temperature and mass ratio within a suitable range is beneficial to the full dispersion of dermal tissue particles, and the first mixed reagent can quickly penetrate deep into the skin to remove impurities without damaging the natural collagen fiber structure.

[0020] In some embodiments, in step S1, the reaction time of the dermal tissue particles and the second mixed reagent is 4h to 6h, the temperature is 25℃ to 35℃, and the mass ratio of the dermal tissue particles to the first mixed reagent is 1:(2~3).

[0021] In the above technical solution, adjusting the reaction time, temperature and mass ratio within a suitable range is beneficial to the full dispersion of dermal tissue particles after swelling and the rapid diffusion of the second mixed reagent, thereby achieving efficient shrinkage without damaging the natural collagen fiber structure.

[0022] Furthermore, after mixing and reacting the dermal tissue particles with the first mixing reagent, the process further includes: filtration and washing. Furthermore, after mixing and reacting the dermal tissue particles with the second mixing reagent, the process further includes: filtration, washing, and centrifugation.

[0023] In some embodiments, the method for preparing dermal tissue particles includes: freezing and thawing mammalian skin, then removing hair and defatting to obtain the dermis; cutting the dermis to obtain dermal tissue particles.

[0024] In the above technical solution, by freezing and thawing at low temperatures, the dense microstructure of animal dermal tissue can be gently disrupted without damaging the structure of the collagen fibers themselves. Water molecules freeze into ice crystals and expand during the freezing process, thereby generating mechanical pressure that causes the cell membrane to rupture. The fragmented cells help to dissolve nucleic acid substances and remove cells between collagen fibers in subsequent processing, which is conducive to further improving the protein purity and extraction rate of natural collagen fibers.

[0025] In some implementations, the length of the dermal tissue particles is 1 mm to 5 mm.

[0026] In the above technical solution, the length of the dermal tissue particles is within a small range, which is conducive to uniform dispersion and reaction in subsequent processing, reduces entanglement and aggregation, and further improves the dispersibility of natural collagen fibers.

[0027] In some embodiments, step S2 includes: freezing the impurity-removed collagen particles in liquid nitrogen, and then subjecting them to cryogenic crushing and cutting at -80°C to -120°C to obtain short collagen fibers.

[0028] In the above technical solution, collagen fibers become brittle under the ultra-low temperature environment of liquid nitrogen. Short collagen fibers can be obtained through ultra-low temperature crushing and cutting without affecting the natural structure of natural collagen fibers. Furthermore, the ultra-low temperature pulverization environment can greatly reduce the charge adsorption between collagen fibers, reduce aggregation and entanglement, and effectively improve fiber dispersibility.

[0029] In some implementations, step S2 includes: S21: The impurity-removed collagen particles and the protective solution are mixed and then mechanically crushed to obtain long collagen fibers; wherein, the protective solution includes a citrate-sodium acetate buffer solution with a pH of 5.0~6.0; S22: Long collagen fibers are frozen in liquid nitrogen and then subjected to cryogenic crushing and cutting at -80℃ to -120℃ to obtain short collagen fibers.

[0030] In the aforementioned technical solution, collagen fibers contain abundant charged groups such as -NH2, -COOH, -OH, -SH, and -CONH2, possessing a large amount of amphoteric charge and exhibiting strong electrostatic effects, making them prone to aggregation and entanglement. By adding a protective solution during mechanical crushing, a citrate-sodium acetate buffer solution with an isoelectric point of approximately 5.0 and a pH of 5.0-6.0 can neutralize the charge of the collagen fibers, reducing fiber aggregation caused by electrostatic adsorption and charge attraction, thereby effectively improving the dispersibility and uniformity of natural collagen fibers. Further cryogenic crushing and cutting of long collagen fibers using liquid nitrogen freezing can significantly reduce the charge adsorption between collagen fibers, minimizing aggregation and entanglement, and effectively improving fiber dispersibility.

[0031] Furthermore, the mass ratio of impurity-removing collagen particles to protective solution is 1:(0.5~1). A smaller amount of protective solution can effectively encapsulate the impurity-removing collagen particles, reducing their floating and loss, which is beneficial for improving grinding efficiency and further increasing the extraction rate.

[0032] Furthermore, the protective solution also includes monohydric alcohol, which accounts for 10% to 20% of the total mass. Adding a certain amount of monohydric alcohol to the protective solution can cause mild dehydration, inhibit collagen swelling, maintain a firm structure, and facilitate breaking and cutting, thereby ensuring the dispersibility of collagen fibers.

[0033] Furthermore, the mechanical crushing speed is 2000 rpm to 5000 rpm, and the time is 1 min to 2 min. By controlling the process parameters of mechanical crushing within a suitable range, it is beneficial to gently dissociate the impurity-removed collagen particles into long collagen fibers, and also to improve the dispersibility and uniformity of the fibers.

[0034] Furthermore, in step S22, the collagen long fibers and the protective solution are mixed and then frozen in liquid nitrogen. By mixing the protective solution, it is beneficial to further reduce fiber aggregation and improve fiber dispersibility during the liquid nitrogen freezing process.

[0035] Furthermore, the length of the collagen long fibers is 1mm to 5mm. Using the aforementioned mechanical grinding method, impurity-free collagen particles can be ground into millimeter-sized collagen long fibers, maintaining good dispersibility.

[0036] In some implementations, the length of the short collagen fibers is less than or equal to 850 μm.

[0037] In the above technical solution, the length of collagen fibers reaches the micrometer level, and the entanglement and aggregation between fibers are greatly reduced, which can significantly improve their dispersibility and processability, and is conducive to the further industrial application of natural collagen fibers.

[0038] In some embodiments, step S21 is followed by: enzymatic hydrolysis of the collagen long fibers at a pH of 6 to 8.

[0039] In the above technical solution, further enzymatic hydrolysis under a neutral environment can remove telopeptides and miscellaneous protein adhesions between collagen fibers without damaging the collagen backbone and triple helix structure, thus preserving the fiber's natural structure. Furthermore, by controlling the neutral environment, the fibers do not swell secondary, which helps maintain their tightness, rigidity, and morphological integrity, and promotes the non-entanglement, non-aggregation, and dispersion of collagen fibers.

[0040] Furthermore, the enzymes used in the enzymatic hydrolysis include at least one of bromelain, trypsin, or trypsin. Bromelain, trypsin, or trypsin all exhibit high activity in a neutral pH environment and can effectively hydrolyze telopeptides and contaminating proteins in long collagen fibers at a pH of 6-8 without inducing secondary swelling.

[0041] Furthermore, adding a protective solution during the enzymatic hydrolysis process can effectively stabilize the pH of the enzymatic hydrolysis reaction system, improve the efficiency and uniformity of enzymatic hydrolysis, and also help maintain the good dispersibility of collagen long fibers.

[0042] Furthermore, the mass ratio of collagen long fibers to enzyme is (200:1) to (1:50), the enzymatic hydrolysis temperature is 4℃ to 25℃, and the time is 12h to 24h. Controlling the enzymatic hydrolysis process parameters can further improve the efficiency of telopeptide removal, reduce impurity residue, and protect the natural structure of natural collagen fibers from damage.

[0043] In some embodiments, step S2 is followed by: mixing collagen short fibers with water to obtain a slurry; and vacuum spray drying the slurry at 40°C to 55°C.

[0044] In the above technical solution, by further low-temperature vacuum drying, fluffy natural collagen fibers are obtained, which is beneficial to further improve dispersibility, rehydration and processing performance, and retains the natural collagen structure of the fibers.

[0045] Furthermore, the solid content of the slurry is 1.0%~5.0%, which is beneficial for the uniform and sufficient dispersion of collagen fibers and rapid dehydration and drying.

[0046] Secondly, embodiments of this application also provide a natural collagen fiber, which is prepared using the preparation method provided in the first aspect of this application.

[0047] In the above-mentioned technical solution, the natural collagen fiber provided in this application is an insoluble collagen fiber with strong fibrous properties, an intact natural fiber structure, a superhelical quadrilateral structure, exhibiting alternating light and dark stripes, and good retention of type I and type III collagen components. It has low immunogenicity, low levels of impurities such as fat, cells, and extraneous proteins, and a collagen purity of over 99.5%. Furthermore, the collagen fibers exhibit weak electrostatic adsorption, good dispersibility and stability, and are easy to process into downstream products using this as a raw material.

[0048] Thirdly, embodiments of this application also provide the application of the natural collagen fibers provided in the second aspect in the preparation of repair materials, hemostatic materials, tissue engineering materials, or medical filler materials.

[0049] In the above technical solution, the natural fibrous adhesive raw material provided in this application meets the requirements of medical standards. It has the characteristics of strong fibrousness, complete natural fiber structure, easy dispersion and uniformity, and easy processing. It can be effectively used to prepare repair materials (such as collagen sponges, collagen membranes, collagen powder), hemostatic materials, tissue engineering materials, medical filler materials, etc. The prepared products have good rehydration, mechanical properties, thermal stability, degradation resistance and bioactivity. Attached Figure Description

[0050] To more clearly illustrate the technical solutions of the embodiments of this application, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of this application and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0051] Figure 1 A process flow diagram of a method for preparing natural collagen fibers provided in one embodiment of this application.

[0052] Figure 2 These are optical images of the natural collagen fibers prepared in Examples 1, 3 and 5 of this application.

[0053] Figure 3 This is a microscope image of the natural collagen fibers prepared in Example 1 of this application.

[0054] Figure 4 This is a microscope image of the natural collagen fibers prepared in Example 3 of this application.

[0055] Figure 5 This is a microscope image of the natural collagen fibers prepared in Example 5 of this application.

[0056] Figure 6 This is a SEM image of the natural collagen fibers prepared in Example 1 of this application.

[0057] Figure 7 This is a microscopic image of natural scarlet staining of the natural collagen fibers prepared in Example 1 of this application.

[0058] Figure 8 This is a microscopic image of natural scarlet staining of the natural collagen fibers prepared in Example 3 of this application.

[0059] Figure 9 This is a microscopic image of natural scarlet staining of the natural collagen fibers prepared in Example 5 of this application.

[0060] Figure 10 This is an optical image of the collagen membrane prepared in Example 1 of this application.

[0061] Figure 11 This is a DSC heat flow-temperature curve of the collagen membrane prepared in Example 1 of this application. Detailed Implementation

[0062] The following detailed description, with appropriate reference to the accompanying drawings, discloses the natural collagen fibers, methods for preparing the natural collagen fibers, and embodiments thereof, but some unnecessary details may be omitted. For example, detailed descriptions of well-known matters and repetitive descriptions of actually identical structures may be omitted. This is to avoid unnecessarily lengthy descriptions and to facilitate understanding by those skilled in the art. Furthermore, the accompanying drawings and the following description are provided for those skilled in the art to fully understand this application and are not intended to limit the subject matter of the claims.

[0063] The "range" disclosed in this application is defined by a lower limit and an upper limit. A given range is defined by selecting a lower limit and an upper limit, which define the boundaries of a particular range. Ranges defined in this way can include or exclude endpoints and can be arbitrarily combined; that is, any lower limit can be combined with any upper limit to form a range. For example, if ranges of 60~120 and 80~110 are listed for a specific parameter, it is also expected that ranges of 60~110 and 80~120 are also included. Furthermore, if minimum range values ​​of 1 and 2 are listed, and if maximum range values ​​of 3, 4, and 5 are listed, then the following ranges are all expected: 1~3, 1~4, 1~5, 2~3, 2~4, and 2~5. In this application, unless otherwise stated, the numerical range "a~b" represents a shortened representation of any combination of real numbers between a and b, where a and b are real numbers. For example, the numerical range "0~5" indicates that all real numbers between "0~5" have been listed in this article; "0~5" is simply a shortened representation of these numerical combinations. Furthermore, when a parameter is stated as an integer ≥2, it is equivalent to disclosing that the parameter is, for example, an integer such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, etc.

[0064] Unless otherwise specified, all embodiments and optional embodiments of this application can be combined to form new technical solutions.

[0065] Based on differences in solubility, natural collagen fibers can be divided into soluble collagen fibers and insoluble collagen fibers. Soluble collagen generally refers to single collagen molecules that do not possess a supramolecular structure. Although they retain a triple helix structure, the natural fiber structure has been destroyed. In contrast, insoluble collagen fibers (i.e., natural collagen fibers) possess a more complete supramolecular fiber structure, triple helix structure, and a pure natural fiber morphology. They retain natural active groups and excellent mechanical properties, degradation resistance, and acid and alkali resistance to the greatest extent. Furthermore, due to their dense triple helix structure and interwoven supramolecular structure, they can form multi-level fibrous structures ranging from nanometer to micrometer and millimeter scales. They can even aggregate into macroscopic, longer, and thicker fiber bundles, giving them hydrophilic but water-insoluble properties, as well as a certain degree of resistance to acids and alkalis. All of these factors allow insoluble collagen fibers to retain excellent plasticity and processing performance during subsequent product processing, making them ideal medical materials widely used in various clinical fields such as wound repair, hemostasis, bone tissue regeneration, soft tissue filling, and implantation.

[0066] However, existing research mainly focuses on soluble collagens such as acid-soluble, alkali-soluble, and salt-soluble collagens as the main products. These methods require strong acid, strong alkali, or high salt conditions to destroy the natural aggregation structure of collagen. This not only leads to the disintegration of the supramolecular structure of collagen fibers and the loss of the unique advantages of natural collagen fibers, but also has problems such as the loss of type I and type III collagen components and low extraction rates, making it difficult to meet the preparation requirements of high-performance medical collagen raw materials.

[0067] Therefore, researching a preparation method to obtain high-purity, high-extraction-rate insoluble collagen fibers without destroying the natural aggregate structure of collagen and retaining the components of type I and type III collagen is of significant research value. Furthermore, because collagen fibers contain abundant charged groups such as -NH2, -COOH, -OH, -SH, and -CONH2, they possess a large number of amphoteric charges and exhibit strong electrostatic interactions. Overcoming electrostatic adsorption to prepare well-dispersible, non-aggregated, and non-entangled natural collagen fibers is also a key research focus.

[0068] Based on this, this application provides a method for preparing natural collagen fibers. The method involves sequentially treating dermal tissue particles with a first and a second mixing reagent to obtain purified collagen particles. These purified collagen particles are then pulverized to obtain insoluble natural collagen fibers. This method effectively preserves the natural morphology of the collagen fibers. The product exhibits a superhelical quaternary structure formed by the aggregation of natural triple helical structures, displaying alternating light and dark stripes. Furthermore, it effectively retains type I and type III collagen components, exhibits low immunogenicity, and has low levels of impurities such as fat, cells, and other proteins, with a collagen purity exceeding 99.5%. In addition, this preparation method is simple, efficient, and has an extraction rate exceeding 95%, making it easily applicable on a large scale.

[0069] In the first mixed reagent, the strong base-weak acid salt is weakly alkaline. When combined with a low concentration of inorganic alkaline reagent, it causes a gentle swelling of collagen fibers in animal tissues, effectively removing soluble impurities such as fats, interstitial proteins, glycoproteins, and cells. Furthermore, by controlling the low concentrations of the strong base-weak acid salt and inorganic alkaline reagent, the natural triple helix structure of collagen can be avoided, thus preserving type I and type III collagen components well.

[0070] In the second mixed reagent, the higher concentration of monohydric alcohol provides an antibacterial environment and causes dermal tissue particles to shrink and collagen fibers to dehydrate, removing impurities dissolved during the swelling process without damaging the collagen fiber structure. Furthermore, the swollen collagen fibers rapidly shrink and compact, forming tight, full, impurity-free collagen particles, effectively preventing fiber entanglement and aggregation during subsequent pulverization and improving the dispersibility of natural collagen fibers in subsequent processing.

[0071] The following description, in conjunction with the accompanying drawings, details the natural collagen fibers, their preparation methods, and applications according to embodiments of this application.

[0072] Figure 1 For a process flow diagram of a method for preparing natural collagen fibers provided in this application embodiment, please refer to [link / reference]. Figure 1 The preparation method includes the following steps: S1: Dermal tissue particles derived from mammals are sequentially mixed with a first mixing reagent and a second mixing reagent to obtain impurity-free collagen particles; wherein, based on the total mass of the first mixing reagent, the first mixing reagent includes water, a strong base-weak acid salt at a mass percentage of 1% to 3%, and an inorganic base reagent at a mass percentage of 0.15% to 0.8%; based on the total mass of the second mixing reagent, the second mixing reagent includes water and a monohydric alcohol at a mass percentage of 40% to 50%.

[0073] As an example, in the first mixed reagent, the mass percentage of the strong base-weak acid salt is 1%, 1.5%, 2%, 2.5%, 3%, etc.; the mass percentage of the inorganic base reagent is 0.15%, 0.3%, 0.5%, 0.6%, 0.8%, etc. In the second mixed reagent, the mass percentage of the monohydric alcohol is 40%, 42%, 45%, 48%, 50%, etc.

[0074] In some embodiments, the strong base weak acid salt includes at least one of sodium bicarbonate (NaHCO3), sodium carbonate (Na2CO3), or sodium acetate (CH3COONa); the inorganic base reagent includes at least one of sodium hydroxide (NaOH), potassium hydroxide (KOH), or calcium hydroxide (Ca(OH)2); and the monohydric alcohol includes at least one of ethanol, n-propanol, isopropanol, or isobutanol.

[0075] In some embodiments, the first mixed reagent further includes an oxidant at a mass percentage of 0.5% to 1.0%. For example, the mass percentage of the oxidant is 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, etc.

[0076] Furthermore, the oxidant includes at least one of hydrogen peroxide (H2O2) or sodium hypochlorite (NaClO).

[0077] In some embodiments, the first mixed reagent further includes a surfactant at a mass percentage of 0.01% to 0.05%. For example, the surfactant in the first mixed reagent may be at a mass percentage of 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, etc.

[0078] Furthermore, the surfactant includes at least one of anionic or cationic surfactants. For example, the anionic surfactant includes sodium dodecylbenzenesulfonate (SDS); the cationic surfactant includes hexadecylpyridine chloride.

[0079] In some embodiments, the second mixed reagent further includes a metal ion chelating agent at a mass percentage of 0.5% to 1.0%. For example, the mass percentage of the metal ion chelating agent in the second mixed reagent is 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1.0%, etc.

[0080] Further, the metal ion chelating agent includes at least one of disodium ethylenediaminetetraacetate or tetrasodium ethylenediaminetetraacetate. Preferably, the metal ion chelating agent includes disodium ethylenediaminetetraacetate.

[0081] In some embodiments, the second mixture further includes an astringent at a mass percentage of 0.1% to 0.5%; the astringent includes organic acid compounds and / or polyphenolic compounds. For example, the astringent in the second mixture is at a mass percentage of 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, etc.

[0082] Furthermore, the astringent includes at least one of tannic acid, catechin, or tea polyphenols. Preferably, the astringent includes tannic acid.

[0083] In some embodiments, the reaction time between the dermal tissue particles and the first mixed reagent is 8h to 12h, the temperature is 25℃ to 35℃, and the mass ratio of the dermal tissue particles to the first mixed reagent is 1:(2 to 5). As examples, the reaction time is 8h, 9h, 10h, 11h, 12h, etc.; the temperature is 25℃, 30℃, 35℃, etc.; and the mass ratio is 1:2, 1:3, 1:4, 1:5, etc.

[0084] Furthermore, after mixing and reacting the dermal tissue particles with the first mixing reagent, the process also includes filtration and washing. As an example, purified water is used to wash away any residual first mixing reagent after filtration.

[0085] In some embodiments, the reaction time between the dermal tissue particles and the second mixed reagent is 4 h to 6 h, the temperature is 25 °C to 35 °C, and the mass ratio of the dermal tissue particles to the first mixed reagent is 1:(2 to 3). As examples, the reaction time is 4 h, 5 h, 6 h, etc.; the temperature is 25 °C, 30 °C, 35 °C, etc.; and the mass ratio is 1:2, 1:2.5, 1:3, etc.

[0086] Furthermore, after mixing and reacting the dermal tissue particles with the second reagent, the process also includes filtration, washing, and centrifugation. Washing can begin with a 5%–10% glycerol aqueous solution, followed by washing with purified water. Centrifugation can be performed at 1000–2000 rpm; high-speed centrifugation effectively removes any residual reaction liquid.

[0087] In some embodiments, the method for preparing dermal tissue particles includes: The dermis is obtained by freezing and thawing mammal skin, followed by hair removal and degreasing. The dermis is cut to obtain dermal tissue particles.

[0088] Understandably, freezing and thawing can be done once or repeatedly in a cycle.

[0089] In some embodiments, mammals may include pigs, cattle, sheep, horses, deer, etc. Among these, because the collagen fibers in the dermal tissue of young animals have a low degree of cross-linking, a considerable portion of the collagen fibers constituting the dermal network structure are in a pre-collagen fiber state, relatively dispersed, interspersed, and with more consistent fiber orientation. Furthermore, the content of type III collagen is relatively higher in the dermal tissue of adult animals, making it more conducive to the extraction of natural collagen fibers. Therefore, mammals preferably include fetal cattle or sheep within one month of birth, or pigs within six months of birth.

[0090] It should be noted that all mammalian sources used in this application comply with the medical standard "YY / T 0771.2-2020 Animal-derived Medical Devices Part 2: Control of Source, Collection and Disposal". The dermis is preferably treated until there is no fat or necrotic tissue on the fleshy surface, and the skin surface and hair roots are thoroughly removed to facilitate subsequent reactions.

[0091] Furthermore, the thickness of the dermis can be 1mm to 5mm, such as 1mm, 2mm, 3mm, 4mm, 5mm, etc.

[0092] In some embodiments, the length of the dermal tissue particles is 1mm to 5mm, such as 1mm, 2mm, 3mm, 4mm, 5mm, etc.

[0093] S2: Crush the impurity-removed collagen particles to obtain natural collagen fibers.

[0094] Understandably, conventional techniques in the field can be used to pulverize and dissociate impurity-removed collagen particles into natural collagen fibers, such as mechanical crushing, low-temperature crushing, and ultrasonic crushing.

[0095] In some embodiments, step S2 includes: freezing the impurity-removed collagen particles in liquid nitrogen, and then performing cryogenic crushing and cutting at -80°C to -120°C to obtain collagen short fibers.

[0096] The cryogenic pulverization step can be performed using a liquid nitrogen pulverizer. First, the equipment hopper is pre-cooled to -80℃ to -120℃. The material frozen by liquid nitrogen is then fed into the hopper. The gap of the toothed ring in the pulverizing chamber is set to medium to large, and the rotation speed is adjusted to 4000rpm to 5000rpm. When the frozen material passes through the high-speed rotating blades, it collides and cuts with the cutting edge, which can untangle the fibers and cut them into short fibers. The crushed material is discharged to the collection device through a cold air blower.

[0097] In some embodiments, step S2 includes: S21: The impurity-removed collagen particles and the protective solution are mixed and then mechanically crushed to obtain long collagen fibers; wherein, the protective solution includes a citrate-sodium acetate buffer solution with a pH of 5.0~6.0; S22: Long collagen fibers are frozen in liquid nitrogen and then subjected to cryogenic crushing and cutting at -80℃ to -120℃ to obtain short collagen fibers.

[0098] In this application, the citrate-sodium acetate buffer solution can be prepared using citric acid, sodium acetate, and water according to conventional methods. The total molar concentration (sodium ion concentration) of the citrate-sodium acetate buffer solution can be 0.05 mol / L to 0.1 mol / L.

[0099] Furthermore, the mass ratio of the impurity-removing collagen particles to the protective solution is 1:(0.5~1). As examples, the mass ratio of the impurity-removing collagen particles to the protective solution is 1:0.5, 1:0.6, 1:0.7, 1:0.8, 1:0.9, 1:1, etc.

[0100] Furthermore, the protective solution also includes a monohydric alcohol, which accounts for 10% to 20% of the total mass of the protective solution. For example, the mass percentage of the monohydric alcohol in the protective solution is 10%, 12%, 15%, 16%, 18%, 20%, etc. Even further, the monohydric alcohol may include at least one of ethanol, n-propanol, isopropanol, or isobutanol.

[0101] In some embodiments, the length of the collagen fibers is 1 mm to 5 mm. As an example, the length of the collagen fibers is 1 mm, 2 mm, 3 mm, 4 mm, 5 mm, etc.

[0102] In some embodiments, mechanical crushing can be carried out using mechanical equipment such as a roller mill or colloid mill, with a rotation speed of 2000 rpm to 5000 rpm and a time of 1 min to 2 min.

[0103] In some embodiments, the cryogenic crushing and cutting steps can be performed using a liquid nitrogen crusher. The equipment hopper is first pre-cooled to -80°C to -120°C. The material frozen by liquid nitrogen is then introduced into the hopper. The gap of the crushing chamber tooth ring is set to medium to large, and the rotation speed is adjusted to 4000 rpm to 5000 rpm. When the frozen material passes through the high-speed rotating blades, it collides and cuts with the cutting edge, which can untangle the fibers and cut them into short fibers. The crushed material is discharged to the collection device through a cold air blower.

[0104] In some embodiments, step S21 is followed by: enzymatic hydrolysis of the collagen long fibers at a pH of 6 to 8.

[0105] Furthermore, the enzymes used in the enzymatic hydrolysis process include at least one of bromelain, trypsin, or trypsin.

[0106] Furthermore, a protective solution can be added during the enzymatic hydrolysis process.

[0107] Furthermore, the mass ratio of collagen long fibers to enzyme is (200:1) to (1:50), the enzymatic hydrolysis temperature is 4℃ to 25℃, and the time is 12h to 24h. As examples, the mass ratio of collagen long fibers to enzyme is 200:1, 100:1, 50:1, 1:1, 1:50, etc.; the enzymatic hydrolysis temperature is 4℃, 10℃, 15℃, 20℃, 25℃, etc.; and the time is 12h, 15h, 18h, 20h, 24h, etc.

[0108] It should be noted that since the collagen fibers and protective solution are mechanically crushed after mixing to form a slurry, the mass ratio of collagen fibers to enzymes is calculated using the mass of the wet collagen fibers, not the dry weight.

[0109] Furthermore, the length of the collagen short fibers is less than or equal to 850 μm, preferably 50 μm to 500 μm. As examples, the length of the collagen short fibers can be 100 μm to 800 μm, 50 μm to 300 μm, 50 μm to 250 μm, 100 μm to 500 μm, etc.

[0110] In some embodiments, after step S2, the method further includes: mixing collagen short fibers with water to obtain a slurry; and vacuum spray drying the slurry at a temperature of 40°C to 55°C. Further, the inlet air temperature of the vacuum spray dryer can be set to 50°C to 55°C, and the outlet air temperature can be set to 40°C to 45°C.

[0111] Further, the solid content of the slurry is 1.0%~5.0%, for example, 1.0%, 2.0%, 3.0%, 4.0%, 5.0%, etc. Additionally, this application embodiment also provides a natural collagen fiber prepared using the above-described preparation method. This natural collagen fiber is an insoluble collagen fiber, with strong fibrous properties, an intact natural fiber structure, a superhelical quadrilateral structure, and can exhibit alternating light and dark stripes. It also retains type I and type III collagen components well, has low immunogenicity, low levels of impurities such as fat, cells, and extraneous proteins, and a collagen purity of over 99.5%. Furthermore, the electrostatic adsorption between collagen fibers is weak, resulting in good dispersibility, stability, and ease of processing.

[0112] In addition, embodiments of this application also provide the application of the above-mentioned natural collagen fibers in the preparation of repair materials, hemostatic materials, tissue engineering materials or medical filler materials.

[0113] This natural fibrous gel raw material meets medical standards and features strong fibrous properties, intact natural fiber structure, easy dispersion and uniformity, and easy processing. It can be effectively used to prepare repair materials (such as collagen sponges, collagen membranes, and collagen powder), hemostatic materials, tissue engineering materials, and medical filler materials. The prepared products have excellent rehydration properties, mechanical properties, thermal stability, degradation resistance, and high bioactivity.

[0114] Among them, the collagen membrane prepared from this natural collagen fiber has high tensile strength, flexibility, rehydration, stability and enzyme hydrolysis tolerance without cross-linking reaction.

[0115] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0116] Example 1 This embodiment provides a natural collagen fiber, the preparation method of which includes the following steps: 1. Preparation of dermal tissue particles 1.1 Select fetal calves within one month of age, whose sources meet the medical standard "YY / T 0771.2-2020 Animal-derived Medical Devices Part 2: Control of Source, Collection and Disposal". After mechanically peeling off the fur, freeze it for later use. Take 25 kg of frozen fur and thaw it. First, use a shearing tool to remove most of the hair. Then, use a meating machine to remove the fat and muscle tissue from the meat surface. Next, use a precision skinning machine with a skinning thickness of approximately 0.8 mm to remove as much residue as possible from the meat surface. Then, use a skinning machine with a skinning thickness of 1.0 mm to remove the hair, hair follicles, and hair roots from the fur surface, obtaining a clean dermis layer, which is then frozen and stored for later use.

[0117] 1.2 Remove the clean dermis layer and cut it with a three-dimensional dicing machine with a blade-to-cutting gap of 2mm. A small number of dermis tissue particles larger than 2mm are cut again with a dicing machine with a blade-to-cutting gap of 2mm until dermis tissue particles of about 2mm in size are obtained. Then, wash them repeatedly with a sodium hypochlorite-purified aqueous solution of 0.1% by mass to obtain clean dermis tissue particles.

[0118] 2. Preparation of impurity-free collagen particles 2.1 Take 10 kg of washed dermal tissue particles and add 20 kg of the first mixing reagent. The first mixing reagent includes: 0.6 kg sodium bicarbonate (3% by mass), 0.1 kg hydrogen peroxide (0.5% by mass), 0.12 kg sodium hydroxide (0.6% by mass), 10 g sodium dodecylbenzenesulfonate (SDS, 0.05% by mass), and 19.17 kg purified water. Control the reaction conditions to react at a solution temperature of 35℃ for 12 h. After the reaction is completed, filter with a screen, then wash twice with 30 kg of purified water, and filter again to obtain primary impurity-removed collagen particles.

[0119] 2.2 Add 30 kg of a second mixed reagent to the above-mentioned primary impurity-removed collagen particles. The second mixed reagent includes: 12 kg of ethanol (40% by mass), 0.3 kg of disodium ethylenediaminetetraacetate (1% by mass), 0.15 kg of tannic acid (0.5% by mass), and 17.55 kg of purified water. Control the reaction conditions to react at a solution temperature of 35℃ for 6 hours. After the reaction, filter the mixture through a screen, wash it once with 5% glycerol solution, and then wash it thoroughly with 30 kg of purified water. Finally, centrifuge the material for 10 minutes at a speed of 1000 rpm, and collect the impurity-removed collagen particles without any washing liquid residue.

[0120] 3. Preparation of long collagen fibers 3.1 First, prepare a 0.05M citrate-sodium acetate buffer solution with a pH of 5.5. Weigh 8.5 kg of the buffer solution and add 1.5 kg of isopropanol, mixing thoroughly to obtain 10 kg of protective solution. Slowly add the protective solution to 10 kg of purified collagen particles, stir and mix well, and let it stand for 1 hour. Then, while stirring, feed the material into the hopper of a colloid mill and grind it at 2000 rpm for 2 minutes. Collect the collagen long fibers. At this point, the collagen long fibers and the protective solution mix to form a slurry. The mass of the wet collagen long fibers in the slurry is 9.74 kg.

[0121] 3.2 Add 10 kg of protective solution and 100 g of trypsin to the above-mentioned slurry containing protective solution, place it in a mixing device equipped with a temperature control device, set the temperature to 25 °C, and carry out a mixed enzymatic hydrolysis reaction for 12 h. After the reaction, remove the enzyme reagent and small molecules by filtration with a filter screen and air pressure, drain the reaction solution, and then rinse with water to obtain long collagen fibers with telopeptides removed.

[0122] 4. Preparation of short collagen fibers Add 10 kg of protective liquid to the above-mentioned long collagen fibers, mix evenly, and then pre-freeze the material with about 30 kg of liquid nitrogen. Pre-cool the liquid nitrogen pulverizer to -100℃ in advance, adjust the gap of the grinding chamber tooth ring to the middle, set the speed to 4000 rpm, put the pre-frozen material into the liquid nitrogen pulverizer for processing, and collect the material in the material bucket to obtain short collagen fibers.

[0123] Example 2 This embodiment provides a natural collagen fiber, the preparation method of which is largely the same as that in Example 1, except that: Step 1.1 does not involve freezing or thawing (i.e., fresh leather is used).

[0124] Example 3 This embodiment provides a natural collagen fiber, the preparation method of which includes the following steps: 1. Preparation of dermal tissue particles 1.1 Select pigs under six months of age, whose sources meet the medical standard "YY / T 0771.2-2020 Animal-derived Medical Devices Part 2: Control of Source, Collection and Disposal". Collect and freeze the cut pigskin. Take 25 kg of frozen pigskin and thaw it. First, use a hair scraper to remove the pig hair, then use an oiling machine to remove the fat, and finally use a precision skinning machine with a skinning thickness of 0.5 mm to remove as much residual fat as possible. Then, use a skinning machine with a skinning thickness of 1.0 mm to remove the hairy epidermis along with the hair follicles and hair roots, obtaining a clean dermis with minimal residue, which is then frozen for later use.

[0125] 1.2 Remove the clean dermal layer and cut it with a three-dimensional dicing machine with a blade-to-cutting gap of 3mm. A small number of dermal tissue particles larger than 3mm are cut again with a dicing machine with a blade-to-cutting gap of 3mm until dermal tissue particles of 3mm size are obtained. Then, wash them repeatedly with a sodium hypochlorite-purified aqueous solution with a mass fraction of 0.2% to obtain clean dermal tissue particles.

[0126] 2. Preparation of impurity-free collagen particles 2.1 Take 10 kg of washed dermal tissue particles and add 50 kg of the first mixing reagent. The first mixing reagent includes: 0.5 kg sodium bicarbonate (1% by mass), 0.25 kg sodium hypochlorite (0.5% by mass), 0.2 kg potassium hydroxide (0.4% by mass), 10 g hexadecylpyridine (0.02% by mass), and 49.04 kg purified water. Control the reaction conditions to react at a solution temperature of 25℃ for 24 h. After the reaction is completed, filter with a screen, then wash once with 30 kg of purified water, and filter again to obtain primary impurity-removed collagen particles.

[0127] 2.2 Add 20 kg of a second mixed reagent to the above-mentioned primary impurity-removed collagen particles. The second mixed reagent includes: 8 kg of n-propanol (40% by mass), 0.1 kg of disodium ethylenediaminetetraacetate (0.5% by mass), 0.1 kg of tannic acid (0.5% by mass), and 11.8 kg of purified water. Control the reaction conditions to react at a solution temperature of 25℃ for 4 hours. After the reaction, filter the mixture through a screen, wash it once with 10% glycerol solution, and then wash it thoroughly with 30 kg of purified water. Finally, centrifuge the material for 5 minutes at a speed of 2000 rpm, and collect the impurity-removed collagen particles without any washing liquid residue.

[0128] 3. Preparation of long collagen fibers First, prepare a 0.1M citrate-sodium acetate buffer solution with a pH of 6.0. Weigh 4.5 kg of the buffer solution and add 0.5 kg of isopropanol, mixing thoroughly to obtain 5.0 kg of protective solution. Slowly add the protective solution to the purified collagen particles, stir to mix, and let stand for 2 hours. Then, while stirring, pass the purified collagen particles and protective solution into the hopper of a colloid mill and grind at 5000 rpm for 1 minute, repeating the grinding process twice. Collect the long collagen fibers, which, when mixed with the protective solution, form a slurry.

[0129] 4. Preparation of short collagen fibers 4.1 The collagen long fiber slurry was pre-frozen with about 50 kg of liquid nitrogen. The liquid nitrogen pulverizer was pre-cooled to -120°C, the gap of the grinding chamber tooth ring was adjusted to the maximum, and the speed was set to 500 rpm. The pre-frozen material was put into the liquid nitrogen pulverizer for refining. The refined collagen fibers were collected into the material bucket by the blower to obtain dispersed, non-aggregated water-containing short collagen fibers.

[0130] 4.2 The collagen short fibers in a low-temperature frozen state were mixed with purified water to form a slurry with a solid content of 1.0%, and then stirred gently. The mixture was then passed into a vacuum low-temperature spray dryer with an inlet air temperature of 55°C and an outlet air temperature of 45°C. The dried, fluffy, and non-aggregated collagen short fibers were collected.

[0131] Example 4 This embodiment provides a natural collagen fiber, the preparation method of which is largely the same as that in Example 3, except that: In step 2.1, sodium hypochlorite, an oxidizing agent, is not added to the first mixed reagent.

[0132] Example 5 This embodiment provides a natural collagen fiber, the preparation method of which includes the following steps: 1. Preparation of dermal tissue particles 1.1 Select fetal sheep within one month of birth, whose source meets the medical standard "YY / T 0771.2-2020 Animal-derived Medical Devices Part 2: Control of Source, Collection and Disposal". After mechanically peeling the fetal sheep skin, freeze it immediately. Fetal sheep skin is thin, with fine, underdeveloped pores, sparse hair, and a relatively thin skin. Take 30 kg of frozen pelts and thaw them. First, use a shearing tool to remove the wool. Then, use a meating machine to remove the fat and muscle tissue from the meat surface. Next, use a precision skinning machine with a skinning thickness of 0.6 mm to remove as much meat residue as possible. Then, use a skinning machine with a skinning thickness of 0.3 mm to remove the hair and a small amount of hair roots from the wool surface. After processing, the remaining dermis layer thickness is approximately 1.5 mm. Freeze and store for later use.

[0133] 1.2 The clean dermal layer was removed and cut using a three-dimensional dicing machine with a blade-cutting gap of 1.5mm to obtain dermal tissue particles with a size of approximately 1.5mm. The cut dermal tissue particles were washed three times with a sodium hypochlorite-purified aqueous solution with a mass fraction of 0.1% to obtain clean dermal tissue particles.

[0134] 2. Preparation of impurity-free collagen particles 2.1 Take 10 kg of washed dermal tissue particles and add 35 kg of the first mixed reagent, which includes: 1.05 kg sodium acetate (3% by mass), 0.35 kg hydrogen peroxide (1% by mass), 0.06 kg calcium hydroxide (0.17% by mass), 17.5 g sodium dodecylbenzenesulfonate (SDS, 0.05% by mass), and 33.52 kg purified water. Control the reaction conditions to react at a solution temperature of 30℃ for 10 h. After the reaction is completed, filter with a screen, then wash twice with 30 kg of purified water, and filter again to obtain primary impurity-removed collagen particles.

[0135] 2.2 Add 25 kg of a second mixed reagent to the above-mentioned primary impurity-removed collagen particles. The second mixed reagent solution includes: 12.5 kg isopropanol (50% by mass), 0.25 kg disodium ethylenediaminetetraacetate (1% by mass), 0.075 kg tannic acid (0.3% by mass), and 12.175 kg purified water. Control the reaction conditions to react at a solution temperature of 30℃ for 5 hours. After the reaction, filter the solution, wash it once with 7.5% glycerol solution, and then wash it thoroughly with 30 kg of purified water. Finally, centrifuge the material for 7 minutes at a speed of 1500 rpm, and collect the impurity-removed collagen particles without any washing liquid residue.

[0136] 3. Preparation of long collagen fibers 3.1 First, prepare a 0.075M citrate-sodium acetate buffer solution with a pH of 6.0. Weigh 8.75 kg of the buffer solution and add 1.25 kg of isopropanol to it, mixing thoroughly to obtain 10 kg of protective solution. Slowly add the protective solution to the purified collagen particles, stir and mix well, and let it stand for 2 hours. Then, while stirring, feed the material into a tissue homogenizer for grinding. Set the speed to 4000 rpm and the time to 2 minutes, collecting the collagen long fibers. At this point, the collagen long fibers and the protective solution mix to form a slurry. The mass of the wet collagen long fibers in the slurry is 9.82 kg.

[0137] 3.2 Add 10 kg of purified water and 150 g of bromelain to the slurry containing the protective solution, place it in a three-dimensional mixer equipped with a temperature control device, set the temperature to 15℃, and carry out a mixed enzymatic hydrolysis reaction for 24 h. After the reaction, remove the enzyme reagent, protective reagent, and small molecules by filtration using a 50-mesh filter. When the material passes through, purge it with compressed air at a pressure of 0.05 MPa. After the reaction solution is discharged, rinse with 5 times the volume of water to remove impurities, obtaining long collagen fibers with telopeptides removed.

[0138] 4. Preparation of short collagen fibers 4.1 Add 10 kg of protective liquid to the long collagen fibers, mix evenly, and then pre-freeze the material with about 40 kg of liquid nitrogen. Pre-cool the liquid nitrogen pulverizer to -120℃ in advance, adjust the gap of the grinding chamber tooth ring to the maximum, set the speed to 4500 rpm, and put the pre-frozen material into the liquid nitrogen pulverizer for fine processing. The fined collagen fibers are collected into the material bucket by the blower to obtain dispersed, non-aggregated water-containing short collagen fibers.

[0139] 4.2 The collagen short fibers in a low-temperature frozen state were mixed with purified water to form a slurry with a solid content of 2.5%. The mixture was stirred slightly and then passed into a vacuum low-temperature spray dryer with an inlet air temperature of 50°C and an outlet air temperature of 40°C. The dry, fluffy, and non-aggregated collagen short fibers were collected.

[0140] Example 6 This embodiment provides a natural collagen fiber, the preparation method of which is largely the same as that in Example 5, except that: In step 3.1, during the preparation of collagen long fibers, the protective solution is replaced with purified water.

[0141] Comparative Example 1 This comparative example provides a natural collagen fiber, the preparation method of which is largely the same as that in Example 1, except that: Step 2.2 is excluded, meaning that the second mixing reagent is not used for the mixing reaction.

[0142] Comparative Example 2 This comparative example provides a natural collagen fiber, the preparation method of which is largely the same as that in Example 1, except that: Replace sodium bicarbonate with sodium sulfate.

[0143] Test case The natural collagen fibers prepared in Examples 1 to 6 and Comparative Examples 1 to 2 were subjected to performance tests. The test results are shown in Table 1. The specific test methods are as follows: (1) Purity and fat content: The test was conducted in accordance with Appendix A "Determination of Purity of Type I Collagen" and 4.11 "Fat Content" of the medical standard "YY / T 1453-2016 Characterization Method of Type I Collagen for Tissue Engineering Medical Devices".

[0144] (2) DNA residue: The DNA residue was detected according to the standard YY / T 1876-2023 Determination of DNA residue in animal-derived biological materials for tissue engineering medical products: Fluorescent staining method.

[0145] (3) α-gal residue: The test was performed in accordance with the standard YYT 1465.5-2016 Medical Device Immunogenicity Evaluation Method Part 5: Determination of α-Gal Antigen Clearance in Animal-Derived Medical Devices by M86 Antibody.

[0146] (4) Fiber length: Take an appropriate amount of natural collagen fiber, dilute and disperse it with 5% Tween 80 purified aqueous solution, then use a dropper to take an appropriate amount of dispersed collagen solution and drop it onto a glass slide. Add Coomassie brilliant blue staining solution to the drop on the glass slide and stain for 5 min to 10 min. Cover with a coverslip, select an appropriate field of view and magnification of the eyepiece and objective lens, adjust the microscope to focus clearly, and then measure and record the fiber length.

[0147] (5) Type I / III Collagen Content: Sirius red staining specifically binds to collagen. Under polarized light irradiation, type I and type III collagen exhibit different optical rotations and display different colors under a polarized light microscope. Type I collagen appears red, while type III collagen appears dark green. This characteristic was used to conduct Sirius red staining experiments and analyze the images to assess the type and content of collagen. First, natural collagen fiber materials were embedded in paraffin and sectioned, then stained with natural scarlet staining solution. The staining was then observed under a polarized light microscope. Collagen types were distinguished based on color differences, and the images were analyzed using software to calculate the ratio of type I and type III collagen.

[0148] (6) Morphology (SEM): The natural collagen fibers in Example 1 were diluted and dispersed with purified water and freeze-dried. Then, they were cut into samples of a certain size, and the cross-section was subjected to low-temperature gold sputtering treatment. The microstructure and periodic D bands of the collagen fibers were observed under an electron scanning microscope at a certain magnification and an accelerating voltage of 3kV~10kV.

[0149] (7) Dispersibility: Take an appropriate amount of natural collagen fiber and dilute it with purified water at a ratio of 1:1000. Stir it evenly with a glass rod and observe the dispersibility between the fibers with the naked eye to assess whether they agglomerate or entangle.

[0150] (8) Extraction rate: The ratio of the mass of the natural collagen fibers (m1) obtained to the mass of the clean dermal tissue particles (m0), both of which are based on dry weight.

[0151] (9) Total bacterial count: After mixing and dispersing an appropriate amount of natural collagen fiber with sterile water, take 1 mL of the mixed liquid under aseptic conditions and test the total bacterial count using the plate pouring method in the microbial limit test of the Chinese Pharmacopoeia: Microbial Counting Method.

[0152] Table 1 Performance test results of natural collagen fibers

[0153] Note: “—” in Table 1 indicates that the data has not yet been tested.

[0154] Figure 2 These are optical images of the natural collagen fibers prepared in Examples 1, 3, and 5 of this application. Figure 2 As can be seen, natural collagen fibers appear as a fine white powder.

[0155] Figure 3 This is an optical microscope image of the natural collagen fibers prepared in Example 1 of this application. Figure 4 These are microscope images of the natural collagen fibers prepared in Example 3 of this application. Figure 5 These are microscope images of the natural collagen fibers prepared in Example 5 of this application. Figures 3-5 As can be seen from Table 1, natural collagen fibers are uniformly dispersed and relatively short, all below 850 μm, and can be as low as 50 μm.

[0156] Figure 6 This is a SEM image of the natural collagen fibers prepared in Example 1 of this application. Figure 6 As can be seen, natural collagen fibers exhibit a striped structure of alternating light and dark bands (periodic D-bands), which means they have an ordered superhelical quaternary structure generated by the aggregation of natural triple helix structures. This indicates that the natural structure of natural collagen fibers has not been destroyed and has not undergone irreversible denaturation or degradation.

[0157] Figure 7 This is a microscopic image of the natural collagen fibers prepared in Example 1 of this application, stained with natural scarlet. Figure 8 This is a microscopic image of the natural collagen fibers prepared in Example 3 of this application, stained with natural scarlet. Figure 9 This is a microscopic image of the natural scarlet staining of the natural collagen fibers prepared in Example 5 of this application. Combined with... Figures 7-8 As shown in Table 1, the type I collagen (corresponding in red in the figure) and type III collagen (corresponding in dark green in the figure) in the natural collagen fiber are well preserved, and the content of type I collagen is much higher than that of type III collagen, indicating that the composition and ratio of type I collagen and type III collagen are well preserved.

[0158] As can also be seen from Table 1, compared with Comparative Example 1 and Comparative Example 2, the natural collagen fibers prepared in Examples 1 to 6 of this application have low DNA residue and immunogenic substances such as α-gal, the DNA content is far lower than the industry standard of 50 ng / mg, and the purity is not less than 99.5%; and the dispersibility is good, with an extraction rate of more than 95%.

[0159] As can be seen from the comparison between Example 1 and Example 2, freezing and thawing animal tissues can further improve the purity of natural collagen fibers, improve fiber dispersibility, and reduce DNA residues and immunogenic substances such as α-gal residues.

[0160] As can be seen from the comparison between Examples 3 and 4, adding an oxidant to the first mixed reagent, combined with an alkaline swelling system, can further improve the dispersibility of natural collagen fibers.

[0161] A comparison of Examples 5 and 6 shows that adding a protective liquid during the crushing of impurity-removing collagen particles can effectively overcome electrostatic adsorption, reduce fiber aggregation and entanglement, and further improve fiber dispersibility.

[0162] As can be seen from Example 1 and Comparative Example 1, when only the first mixed reagent was used in Comparative Example 1, the purity of the obtained natural collagen fibers was significantly reduced, and the impurity content was significantly increased. Type I and Type III collagen components were damaged, and the extraction rate was also significantly reduced.

[0163] As can be seen from the comparison between Example 1 and Comparative Example 2, the use of strong base and strong acid salt (sodium sulfate) in Comparative Example 2 results in a neutral aqueous solution that cannot form a weak base buffer system with a small amount of inorganic base, which significantly reduces the purity and extraction rate of natural collagen fibers.

[0164] Application examples Collagen membranes were prepared using the natural collagen short fibers obtained in Example 1. The preparation method included the following steps: (1) Purified water, glacial acetic acid and the natural collagen short fibers prepared in Example 1 were sequentially added into the stirring homogenizer to make the collagen solid content 8%. The materials were first dispersed by the mechanical stirring device, and then the homogenizer was turned on to further disperse the fibers evenly and mix them evenly with the acid to form a paste-like mixture with a viscosity of 12000cps.

[0165] (2) The above paste-like mixture is extruded through a screw extruder and spread onto a stainless steel plate to a thickness of 0.5 mm. It is then transferred to a forced-air drying oven and dried at 30°C for 4 hours. After that, it is transferred to a vacuum drying oven at 35°C for 2 hours to obtain a smooth, dense collagen film. Figure 10 This is an optical image of a collagen membrane.

[0166] The performance of the above-mentioned collagen membrane was tested using the following methods: (1) Tensile strength: According to the method in ISO 1798:2008 Flexible cellular polymeric materials—Determination of tensile strength and elongation at break, a collagen membrane sample with a width of 15 mm was taken and tested.

[0167] (2) Rehydration: Cut a sample to about 10mm×10mm and place it in physiological saline. Record the time it takes for the sample to fully recover its absorbent state.

[0168] (3) Thermal stability: The test shall be conducted in accordance with the method in Appendix C, Melting Point Determination, of GB / T 1453-2016 Characterization Method for Type I Collagen Sheet Dressings of Tissue Engineering Medical Devices.

[0169] (4) Enzymatic tolerance: Take an appropriate amount of product, place it in a clean test tube, add an appropriate amount of collagenase solution with an enzyme activity of 20 U / ml (prepared with Tris-HCl at pH=7.4), place it at room temperature for enzymatic hydrolysis, observe the degradation of the sample, and record the time for complete enzymatic hydrolysis.

[0170] Tests showed that the tensile strength of the collagen membrane was 1.2 MPa; it also absorbed liquid rapidly upon contact with it, reaching full saturation in 20 seconds. After absorbing liquid, it exhibited good elasticity, full filling, and flexibility, indicating excellent rehydration properties. Figure 11 This is a DSC heat flow-temperature curve of the collagen membrane prepared in Example 1 of this application. Figure 11As can be seen, the thermal denaturation temperature of the collagen membrane is 128.2℃, indicating good thermal stability. Furthermore, in the enzymatic hydrolysis tolerance test, the collagen membrane gradually degraded in the collagenase solution, with a complete degradation time of 9 hours and 16 minutes, demonstrating good enzymatic hydrolysis tolerance. In other words, the collagen membrane sample prepared from natural fibrous collagen in this application exhibits excellent mechanical properties, rehydration properties, and biological properties without any physicochemical cross-linking.

[0171] The embodiments described above are some, but not all, of the embodiments of this application. The detailed description of the embodiments of this application is not intended to limit the scope of the claimed application, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this application without inventive effort are within the scope of protection of this application.

Claims

1. A method for preparing natural collagen fibers, characterized in that, Includes the following steps: S1: Dermal tissue particles derived from mammals are sequentially mixed with a first mixing reagent and a second mixing reagent to obtain impurity-free collagen particles; wherein, based on the total mass of the first mixing reagent, the first mixing reagent includes water, a strong base-weak acid salt at a mass percentage of 1% to 3%, and an inorganic base reagent at a mass percentage of 0.15% to 0.8%; based on the total mass of the second mixing reagent, the second mixing reagent includes water and a monohydric alcohol at a mass percentage of 40% to 50%. S2: Crush the impurity-removed collagen particles to obtain natural collagen fibers.

2. The preparation method according to claim 1, characterized in that, The strong base-weak acid salt includes at least one of sodium bicarbonate, sodium carbonate, or sodium acetate; the inorganic base reagent includes at least one of sodium hydroxide, potassium hydroxide, or calcium hydroxide; the monohydric alcohol includes at least one of ethanol, n-propanol, isopropanol, or isobutanol. And / or, the first mixed reagent further includes an oxidant at a mass percentage of 0.5% to 1.0%; optionally, the oxidant includes at least one of hydrogen peroxide or sodium hypochlorite; And / or, the first mixed reagent further includes a surfactant at a mass percentage of 0.01% to 0.05%; optionally, the surfactant includes anionic surfactants and / or cationic surfactants; And / or, the second mixed reagent further includes a metal ion chelating agent at a mass percentage of 0.5% to 1.0%; optionally, the metal ion chelating agent includes at least one of disodium ethylenediaminetetraacetate or tetrasodium ethylenediaminetetraacetate. And / or, the second mixed reagent further includes an astringent at a mass percentage of 0.1% to 0.5%; the astringent includes organic acid compounds and / or polyphenolic compounds; optionally, the astringent includes at least one of tannic acid, catechin, or tea polyphenols.

3. The preparation method according to claim 1, characterized in that, The reaction time between the dermal tissue particles and the first mixed reagent is 8h~12h, the temperature is 25℃~35℃, and the mass ratio of the dermal tissue particles to the first mixed reagent is 1:(2~5). And / or, the reaction time of the dermal tissue particles with the second mixed reagent is 4h~6h, the temperature is 25℃~35℃, and the mass ratio of the dermal tissue particles to the second mixed reagent is 1:(2~3).

4. The preparation method according to claim 1, characterized in that, The method for preparing the dermal tissue particles includes: freezing and thawing mammalian skin, then removing hair and defatting to obtain the dermal layer; cutting the dermal layer to obtain the dermal tissue particles; Optionally, the length of the dermal tissue particles is 1mm to 5mm.

5. The preparation method according to claim 1, characterized in that, Step S2 includes: freezing the impurity-removed collagen particles in liquid nitrogen, and then performing cryogenic crushing and cutting at -80℃ to -120℃ to obtain collagen short fibers; Optionally, the length of the collagen short fibers is less than or equal to 850 μm.

6. The preparation method according to claim 5, characterized in that, Step S2 includes: S21: The impurity-removed collagen particles and the protective solution are mixed and then mechanically crushed to obtain long collagen fibers; wherein, the protective solution includes a citrate-sodium acetate buffer solution with a pH of 5.0~6.0; S22: The long collagen fibers are frozen in liquid nitrogen, and then subjected to cryogenic crushing and cutting at -80℃ to -120℃ to obtain the short collagen fibers; Optionally, the mass ratio of the impurity-removing collagen particles to the protective liquid is 1:(0.5~1). Optionally, the protective solution further includes a monohydric alcohol, wherein the monohydric alcohol accounts for 10% to 20% by mass in the protective solution; Optionally, the mechanical crushing speed is 2000 rpm to 5000 rpm, and the time is 1 min to 2 min; Optionally, in step S22, the collagen long fibers and the protective solution are mixed and then frozen in liquid nitrogen; Optionally, the length of the collagen fibers is 1mm to 5mm; Optionally, the length of the collagen short fibers is less than or equal to 850 μm.

7. The preparation method according to claim 6, characterized in that, Step S21 is followed by: enzymatic hydrolysis of the collagen long fibers at a pH of 6-8. Optionally, the enzyme used in the enzymatic hydrolysis treatment includes at least one of bromelain, trypsin, or trypsin. Optionally, the protective solution is added during the enzymatic hydrolysis process; Optionally, the mass ratio of the collagen long fibers to the enzyme is (200:1) to (1:50), and the enzymatic hydrolysis treatment is performed at a temperature of 4℃ to 25℃ for 12h to 24h.

8. The preparation method according to any one of claims 5 to 7, characterized in that, Step S2 is followed by: mixing the collagen short fibers with water to obtain a slurry; and vacuum spray drying the slurry at 40°C to 55°C. Optionally, the solid content of the slurry is 1.0% to 5.0%.

9. A natural collagen fiber, characterized in that, It is prepared by the preparation method according to any one of claims 1 to 8.

10. The use of the natural collagen fiber as described in claim 9 in the preparation of repair materials, hemostatic materials, tissue engineering materials or medical filler materials.

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