Method for preparing procollagen from freshwater fish

Abstract

The invention discloses a method for preparing procollagen from freshwater fish. The method comprises the following steps: pressure treatment, physical crushing, mixing emulsification, homogenization, refrigeration, cleaning, extraction, homogenization, inactivation, homogenization and filtration. In the step of mixing and emulsifying, the freshwater fish tissue containing collagen is mixed with a surfactant, so that endotoxin is removed to be lower than 0.25 EU / ml. After adding an enzyme and an acid solution having a pH value of 3 to 6 to the pretreated tissue, the original collagen is extracted. According to the method, the endotoxin can be effectively reduced to be lower than 0.25 EU / ml through pretreatment of the freshwater fish tissue. According to the extraction technology disclosed by the invention, the denaturation temperature of the collagen can be increased while more original collagen is extracted and reserved, and the complete and compact triple-helix structure of the I-type original collagen is kept.

Description

Technical Field

[0001] This invention relates to the preparation of procollagen, and more particularly to a method for removing pigments and endotoxins from collagen-rich freshwater fish tissue and extracting procollagen to meet medical device standards. Background Technology

[0002] Animal skin is composed of 20% to 30% collagen extracellular matrix (ECM). Exposure to ultraviolet (UV) radiation and environmental pollution can lead to collagen loss, causing skin damage, roughness, and aging. In addition, many molecules (such as the MMP enzyme family (matrix metalloproteinases or matrix lysins)) can degrade collagen ECM, making the skin structure unstable and leading to skin aging.

[0003] Most collagen products on the market used in food, cosmetics, skincare products, or pharmaceuticals are collagen fragments. The loose, short-chain structure of these fragments makes them easily broken down by MMP enzymes. Furthermore, due to their incomplete structure, collagen fragments cannot fully induce the genetic response of the collagen ECM. They can only provide a moderate amount of collagen to aging skin and are unlikely to activate the body's ability to repair aging skin.

[0004] Advantages of Type I procollagen: Type I procollagen has been found to effectively protect and repair skin tissue damaged by ultraviolet radiation. In the skin, collagen fibers are formed by the tightly wrapped triple helix structure of Type I procollagen, which promotes the proliferation of fibroblasts and induces the secretion of growth factors to form collagen ECM, thereby protecting the skin from photodamage.

[0005] The density and content of type I procollagen are closely related to skin age. In mature skin, collagen fibers constructed from type I procollagen are looser and shorter, leading to a decrease in the number of fibroblasts and instability of the collagen ECM. In addition, loose collagen fibers are easily broken down into collagen fragments by MMP enzymes, thereby reducing or eliminating their protective ability on the skin. On the other hand, procollagen in the human body can attract fibroblasts to attach and grow, and secrete growth factors to promote the formation of ECM, thereby forming skin protection. Furthermore, the unique structure of procollagen can induce and actively activate the UV signaling pathway for anti-ultraviolet repair.

[0006] In addition, the complete and tightly wound triple helix structure of type I procollagen makes it less susceptible to damage from the external environment, and it is generally more suitable for use in skin care products, medical-grade cosmetics and medical products than collagen fragments. However, with current technology, type I procollagen can only be synthesized and cannot be effectively extracted from animal tissues, and it is difficult to carry out large-scale industrial production, resulting in high market prices.

[0007] Another issue is endotoxin contamination, which is common in collagen materials. This is usually caused by Gram-negative bacteria, which are common in animal habitats and can attach to the skin or be absorbed by the animal's body. During the extraction of procollagen, these bacteria die and release lipopolysaccharide (LPS, also known as endotoxin). Endotoxins can be dangerous when they enter the bloodstream, causing microcirculatory disturbances, septic shock, disseminated intravascular coagulation (DIC), and fever. In addition, the human body is extremely sensitive to the pyrogenic effects of endotoxins. Even injecting trace amounts (1-5 ng / kg body weight) of endotoxin can cause fever and other harm.

[0008] Endotoxins are structurally stable non-protein components that require heating at 250°C for 2 to 4 hours to destroy their activity. Unfortunately, commercially available collagen denatures at temperatures below 100°C, making it difficult to remove endotoxins using the 250°C heating method. Furthermore, while soaking collagen in alcohol and acetone can remove endotoxins, this method typically leads to protein denaturation. Some literature suggests that Triton X-114 cloud point extraction can remove endotoxins, but this method only reduces endotoxin levels from 10 million EU / mL to approximately 100,000 EU / mL, which is still far above the clinically permissible endotoxin limit and may also leave Triton X-114 residues. To reduce endotoxin levels below 0.25 EU / mL, current technologies require specialized column chromatography techniques, which are very expensive and impractical for industrial production.

[0009] Currently, in the industry, to effectively remove endotoxins from collagen materials and meet medical standards, pretreatment with sodium hydroxide (NaOH) is typically involved. However, this method has drawbacks: NaOH can damage protein structure, causing denaturation and preventing optimal protein function. Furthermore, NaOH can reduce the yield of protein extraction. This problem will be demonstrated in the comparative examples that follow in this application.

[0010] Specifically, the treatment time and concentration of NaOH used for tissue processing affect the compactness, length, and denaturation temperature of collagen fibers. Furthermore, excessive use of NaOH in industrial production generates large amounts of waste liquid, causing environmental pollution. On the other hand, the chemical reagents used in this invention to remove pigments and endotoxins from animal tissues, such as urea and sodium chloride (NaCl, salt), can be treated as general wastewater and do not cause environmental burden. Therefore, the tissue processing process in this invention is harmless to the environment.

[0011] Therefore, developing effective methods to remove endotoxins and melanin from animal tissues to medically approved levels and to increase the denaturation temperature of procollagen are problems that need to be overcome in this technical field. The present invention solves this problem. The technology of the present invention does not require the use of NaOH, alcohol or any other chemical liquids that need to be recycled during the removal of endotoxins, and can significantly reduce processing time and energy consumption, providing a novel and environmentally friendly extraction process for large-scale production.

[0012] Therefore, one aspect, advantage, object and embodiment of the present invention is to provide a novel and non-obvious method for industrial-scale production of procollagen.

[0013] Therefore, another aspect, advantage, object, and embodiment of the present invention is to provide a method for extracting procollagen from freshwater fish (including commercially less desirable fish body parts).

[0014] Another aspect, advantage, object, and embodiment of the present invention is to provide a method for extracting procollagen without using NaOH, alcohol, or other contaminants.

[0015] Another aspect, advantage, object, and embodiment of the present invention is to provide a method for producing procollagen that significantly reduces costs compared to synthesis.

[0016] These and other advantages and aspects of the invention will be understood from this application. Summary of the Invention

[0017] This invention provides a method for extracting high-quality, tightly twisted triple-helix collagen from the skin tissue and organs of freshwater fish. The method includes a series of pressure extrusion, mechanical reduction, mixing, and emulsification processes, followed by homogenization, refrigeration, and washing. The next step involves an enzymatic extraction process, followed by multiple rounds of homogenization, combined with enzyme inactivation, and finally filtration.

[0018] The result of this method is a low-cost, high-yield method for producing procollagen while maintaining permissible low endotoxin levels (below the limits required for clinical application).

[0019] The present invention does not require the use of sodium hydroxide (NaOH) and alcohol, and compared with the process using sodium hydroxide, the present invention can produce a very high yield of procollagen (up to 80%, while the yield of the process using sodium hydroxide is 3% or less).

[0020] Therefore, in addition to the above-discussed aspects, advantages, objectives, and embodiments, the present invention provides a method for preparing procollagen from freshwater fish, comprising the following steps:

[0021] (1) Pressure treatment: High pressure is used to remove blood, water and fat from freshwater fish tissue containing collagen;

[0022] (2) Mechanical separation of the freshwater fish tissue; physical crushing: the freshwater fish tissue is chopped and crushed into blocks, strips or powder;

[0023] (3) Mixing and emulsification: The freshwater fish tissue is mixed with surfactant, electrolyte (e.g., Na-X (organic salt)) and urea to obtain an emulsion solution;

[0024] (4) Homogenization: Homogenize the emulsion solution for 13 to 20 minutes;

[0025] (5) Refrigeration: Refrigerate the emulsion solution at 0 to 10°C for 5 to 60 minutes;

[0026] (6) Cleaning: Wash the emulsion solution with water at 38 to 42°C until the absorbance of the cleaning solution at 200 to 300 nm is close to zero, so as to reduce the endotoxin in the freshwater fish tissue to below 0.25 EU / ml;

[0027] (7) Extraction: The freshwater fish tissue is mixed with enzymes and an acidic solution with pH 3-6 to obtain an extract solution;

[0028] (8) Homogenization: Homogenize the extract solution at 4 to 75°C for 2 to 48 hours;

[0029] (9) Inactivation: Add an enzyme inhibitor to the extract solution to lower the pH to below 3;

[0030] (10) Homogenization: The deactivated extract solution is homogenized at 4 to 75°C for 2 to 48 hours; and

[0031] (11) Filtration: The supernatant is separated from the homogenized extract solution by filtration, centrifugation and / or sieving to obtain the desired procollagen.

[0032] Therefore, in addition to the above, one aspect, advantage, object and embodiment of the present invention also includes providing a method for preparing procollagen from freshwater fish, wherein the electrolyte is NaCl and the amount of NaCl added is 1 to 5 wt%.

[0033] Therefore, in addition to the above, one aspect, advantage, object and embodiment of the present invention also includes providing a method for preparing procollagen from freshwater fish, wherein the amount of urea added is 3 to 10 wt%.

[0034] Therefore, in addition to the above, one aspect, advantage, object and embodiment of the present invention also includes providing a method for preparing procollagen from freshwater fish, wherein the surfactant is selected from the group consisting of Tween 20, Tween 80, Triton X-100 and mixtures thereof, and is added in an amount of 0.05 to 0.5 wt%.

[0035] Therefore, in addition to the above, one aspect, advantage, object and embodiment of the present invention also includes providing a method for preparing procollagen from freshwater fish, wherein the activity of the enzyme is between 20 and 2000 U, and the amount of the enzyme added is between 0.5 and 10 wt%.

[0036] Therefore, in addition to the foregoing, one aspect, advantage, object, and embodiment of the present invention also includes providing a method for preparing procollagen from freshwater fish, wherein the enzyme is selected from the group consisting of papain, bromelain, and mixtures thereof.

[0037] Therefore, in addition to the above, one aspect, advantage, object, and embodiment of the present invention also includes providing a method for preparing procollagen from freshwater fish, wherein the acid solution is selected from the group consisting of acetic acid, citric acid, lactic acid, and mixtures thereof, and the amount added is between 0.5 and 1 M.

[0038] Therefore, in addition to the foregoing, one aspect, advantage, object, and embodiment of the present invention also includes providing a method for preparing procollagen from freshwater fish, wherein the enzyme mixture is a combination of bromelain and papain, and the mixing ratio of bromelain and papain is between 0:1 and 1:2.

[0039] Therefore, in addition to the above, one aspect, advantage, object, and embodiment of the present invention also includes providing a method for preparing procollagen from freshwater fish, wherein the enzyme inhibitor is selected from the group consisting of acetic acid, lactic acid, and mixtures thereof, and the mixing ratio of acetic acid and lactic acid is between 1:0 and 1:3.

[0040] Therefore, in addition to the foregoing, one aspect, advantage, object, and embodiment of the present invention also includes providing a method for preparing procollagen from freshwater fish, wherein the electrolyte is an organic salt in the form of Na-X.

[0041] Therefore, in addition to the foregoing, one aspect, advantage, object, and embodiment of the present invention also includes providing a method for preparing procollagen from freshwater fish, wherein the mechanical separation of the freshwater fish tissue further comprises cutting and pulverizing the freshwater fish tissue into a form selected from the group consisting of powder, block, strip, and combinations thereof.

[0042] Therefore, in addition to the above, one aspect, advantage, object, and embodiment of the present invention also includes providing a method for preparing procollagen from freshwater fish, wherein separating the supernatant from the homogenized extract solution further comprises a method selected from the group consisting of filtration, centrifugation, sieving, and combinations thereof. Attached Figure Description

[0043] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the embodiments of the invention to explain the invention and do not constitute a limitation thereof.

[0044] Figure 1 is a flowchart of the method for preparing procollagen according to the present invention;

[0045] Figure 2 A(A), Figure 2 A(B) and Figure 2 A(C) shows macroscopic views of freshwater fish particles before and after the removal of melanin and endotoxins in embodiments and comparative embodiments of the present invention;

[0046] Figure 2 B(A), Figure 2 B(B), Figure 2 B(C) and Figure 2 B(D) shows a macroscopic view of the products (supernatant and precipitate) obtained after extraction of procollagen in the embodiments of the present invention and comparative embodiments;

[0047] Figure 3 A is the Fourier transform infrared (FTIR) spectrum of Embodiment 1 of the present invention;

[0048] Figure 3 B is the FTIR spectrum of Comparative Example 1 of this invention;

[0049] Figure 4 A is the dynamic light scattering (DLS) spectrum of Embodiment 1 of the present invention;

[0050] Figure 4 B is the DLS spectrum of Comparative Example 1 of this invention;

[0051] Figure 5 A is an electrophoresis diagram of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of all embodiments of the present invention;

[0052] Figure 5 B is the SDS-PAGE electrophoresis image of all comparative examples #1 and #2 of this invention;

[0053] Figure 6A is a differential scanning calorimetry (DSC) diagram of Embodiment 1 of the present invention;

[0054] Figure 6B is a DSC diagram of Comparative Example 1 of the present invention;

[0055] Figure 7 is an image of the endotoxin test results of procollagen in Example 1 of the present invention;

[0056] Figure 8 is a schematic diagram of procollagen endothelial stimulation in Example 1 of the present invention;

[0057] Figure 9 is an amino acid composition analysis table of Example 1 of the present invention. Detailed Implementation

[0058] Freshwater fish skin tissue and viscera are rich in collagen. As mentioned earlier, a complete collagen structure plays a significant role in promoting the production and stabilization of collagen ECM in the human body, thereby protecting the skin. This invention selects freshwater fish as the subject, uses live animal tissue as the source of protoplasmic collagen, and applies the technology of this invention to obtain natural protoplasmic collagen that can be used for medical and commercial purposes in an environmentally friendly manner.

[0059] This invention aims to address the shortcomings of existing collagen production technologies by providing an environmentally friendly method that can effectively remove endotoxins and melanin from freshwater fish tissues to medically permissible levels without the use of NaOH, and extract and prepare natural procollagen that is stable, structurally intact, and meets medical standards.

[0060] This invention provides a method for preparing procollagen from freshwater fish, comprising the following steps:

[0061] (1) Pressure treatment: High pressure is used to remove blood, water and fat from freshwater fish tissue containing collagen;

[0062] (2) Physical crushing: The freshwater fish tissue is chopped and crushed into blocks, strips or powder;

[0063] (3) Mixing and emulsification: The freshwater fish tissue is mixed with surfactant, Na-X (such as NaCl) and urea to obtain an emulsion solution;

[0064] (4) Homogenization: Homogenize the emulsion solution for 13 to 20 minutes;

[0065] (5) Refrigeration: Refrigerate the emulsion solution at 0 to 10°C for 5 to 60 minutes;

[0066] (6) Cleaning: Wash the emulsion solution with water at 38 to 42°C until the absorbance of the cleaning solution at 200 to 300 nm is close to zero, so as to reduce the endotoxin in the freshwater fish tissue to below 0.25 EU / ml;

[0067] (7) Extraction: The freshwater fish tissue is mixed with enzymes and an acidic solution with pH 3-6 to obtain an extract solution;

[0068] (8) Homogenization: Homogenize the extract solution at 4 to 75°C for 2 to 48 hours;

[0069] (9) Inactivation: Add an enzyme inhibitor to the extract solution to lower the pH to below 3;

[0070] (10) Homogenization: The deactivated extract solution is homogenized at 4 to 75°C for 2 to 48 hours; and

[0071] (11) Filtration: The supernatant is separated from the homogenized extract solution by filtration, centrifugation and / or sieving to obtain the desired procollagen.

[0072] In one embodiment of the present invention, the amount of NaCl added is 1 to 5 wt%.

[0073] In one embodiment of the present invention, the amount of urea added is 3 to 10 wt%.

[0074] In one embodiment of the present invention, the surfactant is selected from the group consisting of Tween 20, Tween 80, Triton X-100 and mixtures thereof, and is added in an amount of 0.05 to 0.5 wt%.

[0075] In one embodiment of the present invention, the activity of the enzyme is between 20 and 2000 U, and the amount of the enzyme added is between 0.5 and 10 wt%.

[0076] In one embodiment of the present invention, the enzyme is selected from the group consisting of papain, bromelain, and mixtures thereof.

[0077] In one embodiment of the present invention, the acid solution is selected from the group consisting of acetic acid, citric acid, lactic acid and mixtures thereof, and the amount added is between 0.5 and 1 M.

[0078] In one embodiment of the present invention, the enzyme mixture is a combination of bromelain and papain, and the mixing ratio of bromelain and papain is between 0:1 and 1:2.

[0079] In one embodiment of the present invention, the enzyme inhibitor is selected from the group consisting of acetic acid, lactic acid and mixtures thereof, and the mixing ratio of acetic acid and lactic acid is between 1:0 and 1:3.

[0080] One beneficial effect of this invention is that it obtains natural procollagen from freshwater fish without using existing NaOH washing techniques for raw animal tissues. The method of this invention effectively removes pigments and endotoxins to below 0.25 EU / ml, making the treated freshwater fish tissues meet medical standards and suitable for clinical use.

[0081] Furthermore, this invention shortens the preparation process and achieves a yield of 60 to 80% of procollagen. The method for preparing procollagen can increase the denaturation temperature of the processed procollagen and produce procollagen that can be verified as safe through cytotoxicity tests and animal intradermal sensitivity tests. It can be used in medical-grade cosmetics, skin care products, and medical applications.

[0082] Furthermore, the method of the present invention can extract and retain more natural procollagen components, and the procollagen prepared by the present invention has a complete and tightly wound triple helix type I procollagen.

[0083] To further understand the features and technical components of the present invention, please refer to the provided detailed description and drawings. However, the provided drawings are for reference and illustration purposes only and are not intended to limit the scope of the invention.

[0084] Experimental results of implementation and comparison with other methods

[0085] The following specific embodiments illustrate a method for preparing procollagen from freshwater fish according to the present invention. Those skilled in the art can understand the advantages and effects of the present invention from the description of the embodiments. The present invention can be implemented or applied through other different embodiments, and various modifications and changes can be made to the details of the embodiments according to different perspectives and applications without departing from the concept of the present invention. The following embodiments will further describe the relevant technology of the present invention in detail, but the disclosed content is not intended to limit the scope of protection of the present invention.

[0086] It should be noted that although the terms “first,” “second,” “third,” etc., may be used in this document to describe various elements, these elements should not be limited by these terms. These terms are only used to distinguish different elements. Furthermore, as used herein, the word “or” may, depending on the context, include any combination of one or more of the listed items.

[0087] First, please refer to Figure 1. The present invention provides a method for preparing procollagen, comprising the following steps: S101 pressure treatment, S102 physical crushing, S103 mixing and emulsification, S104 homogenization, S105 refrigeration, S106 washing, S201 extraction, S202 homogenization, S203 inactivation, S204 homogenization and S205 filtration.

[0088] Specifically, steps S101 to S106 are pretreatment processes for removing pigments and endotoxins from freshwater fish tissue, and steps S201 to S205 are extraction processes for obtaining procollagen (collagen) components from freshwater fish tissue. These steps are performed in the following order:

[0089] S101 Pressure Processing: Using high pressure to remove blood, water, and fat from freshwater fish tissue containing collagen, which can be fish skin or collagen-rich fish organ tissue.

[0090] S102 Physical crushing: The freshwater fish tissue is chopped and crushed into blocks, strips or powder;

[0091] S103 Mixing and Emulsification: The freshwater fish tissue is mixed with a surfactant, Na-X (such as NaCl), and urea to obtain an emulsion solution.

[0092] Preferably, the surfactant is selected from stock solutions of Tween 20, Tween 80, and Triton X-100, although combinations thereof may also be used. The amount of surfactant added is 0.05 to 0.5 wt%. Preferably, an organic salt is used for NaCl, and the amount of NaCl or other Na-X added may be between 1 and 5 wt%. The amount of urea added is 3 to 10 wt%.

[0093] NaCl can reduce endotoxins because it has the function of regulating osmotic pressure. NaCl increases the osmotic pressure in the extracellular fluid, which may lead to a pressure difference between the extracellular fluid and the intracellular fluid, thereby inhibiting the release of bacterial endotoxins. This is because the high osmotic pressure in the extracellular fluid leads to the loss of intracellular water, causing cell contraction, thereby reducing cell death and the release of endotoxins.

[0094] In addition, NaCl can further reduce the release of endotoxins by inhibiting cell wall breakdown and protease release. The osmotic pressure regulation function of NaCl can reduce the release of bacterial endotoxins and have a positive effect on the body's immune system.

[0095] In addition, other organic salts (such as Na-X) can reduce endotoxin release by promoting the stability of membrane lipids and the strength of the cell wall.

[0096] S104 Homogenization: Homogenize the emulsion solution for 13 to 20 minutes;

[0097] S105 Refrigeration: Refrigerate the emulsion solution at 0 to 10°C for 5 to 60 minutes;

[0098] Ideally, the refrigeration temperature should be 4°C;

[0099] S106 Cleaning: Wash the emulsion solution with water at 38 to 42°C until the absorbance of the washing solution at 200 to 300 nm approaches zero, in order to reduce the endotoxin in the freshwater fish tissue to below 0.25 EU / ml;

[0100] S201 Extraction: Freshwater fish tissue with endotoxin levels below 0.25 EU / ml was mixed with enzymes and an acidic solution with pH 3-6 to obtain an extract solution.

[0101] Specifically, the enzyme activity is between 20 and 2000 U, preferably 2000 U. Furthermore, the enzyme is selected from the group consisting of papain, bromelain, and mixtures thereof, and the amount of the enzyme added is 0.5 to 10 wt%. The acid solution is selected from the group consisting of acetic acid, citric acid, lactic acid, and mixtures thereof, and the amount added is between 0.5 and 1 M.

[0102] Preferably, the enzyme solution is a mixture of bromelain and papain, and the mixing ratio of bromelain to papain is between 0:1 and 1:2.

[0103] S202 Homogenization: The extract solution is homogenized at 4 to 75°C for 2 to 48 hours using methods such as ultrasound, stirring and shaking.

[0104] S203 inactivation: Add an enzyme inhibitor to the extract solution to lower the pH to below 3, preferably below 2.8;

[0105] The enzyme inhibitor is selected from the group consisting of acetic acid, lactic acid and mixtures thereof, and the mixing ratio of acetic acid and lactic acid is between 1:0 and 1:3.

[0106] S204 Homogenization: The inactivated extract solution is homogenized for 2 to 48 hours at 4 to 75°C using methods such as ultrasound, stirring and shaking.

[0107] S205 Filtration: The supernatant is separated from the homogenized extract solution by filtration, centrifugation and / or sieving to obtain the desired procollagen.

[0108] Using the above implementation steps and the concentrations and proportions provided in the formulation composition table of the following examples, the three embodiments of the present invention can be implemented.

[0109] Example 1

[0110] The formulation composition of Example 1 is shown in Table 1.

[0111]

[0112] Example 2

[0113] The formulation composition of Example 2 is shown in Table 2.

[0114] Example 3

[0115] The formulation composition of Example 3 is shown in Table 3.

[0116]

[0117] Comparative Example

[0118] Freshwater fish tissue fragments were prepared and subjected to high-pressure extrusion to remove blood, water, and fat. The fragments were then broken into blocks, strips, or powder by physical cutting and crushing. The freshwater fish tissue particles were then washed with NaOH solution until the endotoxin in the freshwater fish tissue was reduced to below 0.25 EU / ml.

[0119] In Comparative Example 1, particles of freshwater fish tissue were washed with NaOH at 4°C for 24 hours. In Comparative Example 2, particles of freshwater fish tissue were washed with NaOH at 4°C for 30 hours.

[0120] The particles washed with NaOH were then mixed with 0.8M acetic acid and 0.1% by weight of porcine pepsin to obtain an extract. The extract was homogenized at 4°C and then extracted for 240 hours. The supernatant formed after extraction was then collected by filtration, centrifugation and / or sieving to obtain the required proportion of procollagen.

[0121] Comparative Example 1

[0122] The formulation composition of Comparative Example 1 is shown in Table 4.

[0123]

[0124] Comparative Example 2

[0125] Comparative Example 2 was carried out using the implementation steps described in Comparative Example 1, and its formulation was adjusted as shown in Table 5.

[0126]

[0127] Pigment removal effect observation

[0128] In all examples and comparative examples, the freshwater fish tissue fragments were pretreated to remove endotoxins, and the untreated fragments and the pretreated fragments were observed and compared with the naked eye. The untreated fragments and the pretreated fragments from the comparative examples were light gray, while the fragments from the examples were clearly grayish-white, indicating that the examples were more effective than comparative example 1 in removing melanin.

[0129] Please see Figure 2 A. Figure 2 A shows a macroscopic view of the following substances:

[0130] (A) Freshwater fish organ particles containing collagen before pretreatment (sample A);

[0131] (B) Freshwater fish organ particles containing collagen, which were pretreated with surfactant, urea and NaCl and then washed with water (sample B).

[0132] (C) Freshwater fish particles containing collagen that have been pretreated with NaOH.

[0133] Figure 2 B shows a macroscopic view of the following substances:

[0134] (A) The state of sample B after extraction with papain and acetic acid;

[0135] (B) Sample B was extracted with a mixture of bromelain and papain, then treated with a mixture of lactic acid and acetic acid, and then centrifuged to obtain the state of the supernatant and precipitate;

[0136] Freshwater fish skin powder (C) and (D) were pretreated with NaOH and then mixed with acetic acid and porcine pepsin to obtain supernatant (C) and precipitate (D).

[0137] Fourier transform infrared (FTIR) spectroscopy analysis

[0138] Figure 3 A is the FTIR spectrum of procollagen from sample B in this embodiment of the invention, obtained by extraction with a mixture of papain and bromelain, and a mixture of acetic acid and lactic acid.

[0139] like Figure 3 As shown in Figure A, Fourier transform infrared spectroscopy (FTIR) can distinguish several characteristic absorption bands of collagen: amide I is located near 1656 cm⁻¹, amide II is located near 1540 cm⁻¹, and a set of three weaker bands representing the vibrational mode of amide III are concentrated near 1246 cm⁻¹.

[0140] The amide I band originates from the stretching and bending vibrations of the peptide carbonyl (CO) group, appearing as a peak at 1083 cm⁻¹ in the spectrum of fibrous collagen. This peak is slightly shifted from the peaks of amides I and II. Therefore, by comparison... Figure 3 From the spectrum in A, we can identify a specific characteristic peak at 1083 cm⁻¹, which allows us to confirm that the embodiment has a procollagen structure.

[0141] Figure 3 B is the FTIR spectrum of procollagen in the comparative example of this invention. As can be seen from the figure, there is a peak at 3274 cm⁻¹, namely amide A, which is found to be related to… Figure 3 Compared to A, the peak position shifted, and the peak height of amide A was lower. Figure 3The high peak height in A indicates that the alpha helix structure in the comparative example is less stable and has a lower content than that in the examples. The same was observed at amide B, and in addition, there was a small peak at 1080 cm⁻¹, which indicates a lower content of procollagen.

[0142] Intradermal irritation test in animals

[0143] Intradermal irritation tests were conducted on animals according to ISO 10993-1 standard to assess the safety of the extracted procollagen. In the test, polar and non-polar collagen extracts were injected intradermally into the animals, and the presence of any signs of redness, swelling or fever was observed. No such symptoms were observed after 72 hours, and the scores were between 0 and 1.

[0144] Therefore, the embodiments described did not exhibit intradermal irritation. Referring to Figure 8, which is a schematic diagram of procollagen endothelial stimulation analysis (according to ISO 10993-1) in one embodiment of the present invention, the endothelial stimulation data were collected 72 hours after subcutaneous injection of procollagen extracted using a mixture of papain and bromelain and a mixture of acetic acid and lactic acid into New Zealand white rabbits.

[0145] The polarity refers to the procollagen extracted with physiological saline for 24 hours and then subcutaneously injected into New Zealand white rabbits. The polarity control group is physiological saline. The nonpolarity refers to the procollagen extracted with cottonseed oil for 24 hours and then subcutaneously injected into New Zealand white rabbits. The nonpolarity control group is cottonseed oil.

[0146] Triple Helix Feature Test

[0147] The ultraviolet-visible (UV-VIS) absorption spectrum of proteins is mainly determined by the peptide bonds or side chains of the proteins. The amino acid sequence of proteins contains glycine, proline, and hydroxyproline. Triple-helical collagen has a maximum peak around 240 nm.

[0148] Figure 4 A is the dynamic light scattering (DLS) spectrum of procollagen from sample B after extraction with a mixture of bromelain and papain, and a mixture of lactic acid and acetic acid. Figure 4 As shown in Figure A, the full spectrum of Example 1 was detected using dynamic light scattering (DLS). Example 1 showed maximum absorbance near 240 nm and no peaks at other wavelengths, indicating that the triple helix of Example 1 was tightly wound and structurally intact, without producing collagen fragments, thus proving that Example 1 has intact type I procollagen.

[0149] Figure 4 B is the DLS spectrum of collagen in the comparative example, as shown. Figure 4As shown in B, the full spectrum of Comparative Example 1 was detected using DLS. Comparative Example 1 showed maximum absorbance near 240 nm, but the absorbance was not as high as that of Example 1, indicating that the triple helix collagen content in Comparative Example 1 was lower than that in Example 1. In addition, Comparative Example 1 also showed an absorbance peak signal between 200 and 240 nm, indicating that Comparative Example 1 contained many short-wavelength collagen fragments. In other words, compared with Example 1, the purity of triple helix collagen obtained in Comparative Example 1 was lower.

[0150] Sodium dodecyl sulfate polyacrylamide gel electrophoresis

[0151] Figure 5 A shows sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) images of procollagen from Examples 1 (#1), 2 (#2), and 3.

[0152] like Figure 5 As shown in Figure A, the SDS-PAGE results indicate that Example 1 has α1 and α2 chains, indicating that it is type I procollagen. In addition, the γ chain (trimer) also shows the presence of high molecular weight crosslinks.

[0153] Figure 5 B shows the SDS-PAGE electrophoresis image of freshwater fish skin particles:

[0154] (#1) After pretreatment with NaOH for 24 hours, it was then extracted with a mixture of acetic acid and porcine pepsin for 240 hours.

[0155] (#2) After pretreatment with NaOH for 30 hours, it was then extracted with a mixture of acetic acid and porcine pepsin for 240 hours.

[0156] like Figure 5 As shown in Figure B, Comparative Example 1, treated with NaOH for 24 hours, and Comparative Example 2, treated for 30 hours, not only showed α1 and α2 chains and γ chains, but also low molecular weight sequence fragments. Compared to Example 1, the collagen in Comparative Examples 1 and 2 contained numerous low molecular weight sequence fragments, indicating that the triple helix structure of the collagen in Comparative Example 1 failed to retain its properties and was easily degraded into small fragments.

[0157] Cytotoxicity test

[0158] In vitro cytotoxicity assays were performed on mouse fibroblasts (L929) to assess the effect of Example 1 on L929 survival. Cell viability was analyzed by MTT assay according to the cytotoxicity method of ISO 10993-1. After 24, 48 and 72 hours of treatment, the survival rates were approximately 75%, 82% and 88%, respectively, indicating that the procollagen obtained from Example 1 did not exhibit cytotoxicity.

[0159] denaturation temperature

[0160] The denaturation temperature was determined using differential scanning calorimetry (DSC), and the test results are recorded in Table 6 and Figures 6A and 6B. Figure 6A is a DSC image (differential scanning calorimetry image) of procollagen extracted by a mixture of papain and bromelain and a mixture of acetic acid and lactic acid in the embodiments of the present invention. Figure 6B is a DSC image of procollagen in the comparative example.

[0161] As shown in Figure 6A, the denaturation temperature of the embodiment is approximately 77 to 88°C, while the denaturation temperature of the comparative example (see Figure 6B) is approximately 45°C. Therefore, it can be concluded that Embodiment 1 has a more tightly packed / wound triple helix structure.

[0162] Endotoxin content detection

[0163] Endotoxin content was determined using the GEL-CLOTLAL gelation method with Limulus Amebocyte Lysate (LAL) reagent. The LAL reagent used in the test reacts with endotoxin to form a gel block. The presence of endotoxin was detected by visually observing the formation of the gel. The test results of endotoxin content are shown in Table 6 and Figure 7. Figure 7 is an image of the endotoxin test results of procollagen in an embodiment of the present invention (gelation method test).

[0164] (A) Positive control group, endotoxin level greater than 0.25 EU / ml;

[0165] (B) Procollagen extracted from sample B in the examples using a mixture of papain and bromelain and a mixture of acetic acid and lactic acid. (C) Blank group: This sample is free of endotoxin.

[0166] Water protein analysis

[0167] Protein concentration and amino acid sequence were analyzed using LC-MS / MS.

[0168] Figure 9 shows the amino acid composition analysis of procollagen from sample B after extraction with a mixture of papain and bromelain, and a mixture of acetic acid and lactic acid. The amino acid composition analysis results of the comparative collagen are recorded in Table 6. Examples 1 to 3 have high protein concentrations ranging from 1 mg / g to 1.56 mg / g, while the protein concentration of Comparative Example 1 is only 0.2 mg / g. In addition, proline, glycine, glutamine, and arginine are precursors of procollagen. The higher their content, the tighter and more concentrated the triple helix structure of procollagen. As shown in Table 6, the amino acid content of Example 1 is much higher than that of Comparative Example 1.

[0169]

[0170] Beneficial effects of the embodiments

[0171] One beneficial effect of the present invention is that the method of the present invention replaces the existing technology of washing with NaOH, and effectively removes pigments and endotoxins from freshwater fish tissues to below 0.25 EU / ml, so that the treated freshwater fish tissues meet medical standards and can be used in clinical practice.

[0172] Furthermore, this invention shortens the preparation process and achieves a procollagen production yield of over 80%. The procollagen preparation method of this invention can increase the denaturation temperature of pretreated procollagen. Cytotoxicity tests and animal skin sensitivity tests demonstrate its safety for use in skincare, cosmetics, and medical products.

[0173] By using the collagen preparation method provided by this invention, more procollagen components can be extracted and retained, and the prepared procollagen has a complete and tightly wrapped triple helix type I procollagen.

[0174] Another advantage of the method of this invention is that it achieves the goal of maintaining a green environment. All materials used in this invention are "food-grade" materials, and the wastewater discharged during the preparation process is general wastewater, which will not cause environmental pollution or burden. An important aspect of the method of this invention is that it achieves high yield of high-quality procollagen (endotoxin below 0.25 EU / ml) while taking into account environmental protection.

[0175] This invention can be applied to large-scale industrial production to produce high-value procollagen for commercial and medical applications, thus promoting the development of biotechnology.

[0176] The embodiments disclosed above are merely preferred embodiments of the present invention and do not limit the scope of protection of this patent application. Therefore, any equivalent technical changes made using the embodiments and drawings of the present invention are included within the scope of protection of this patent application.

[0177] This disclosure is provided to enable those skilled in the art to implement the invention without unnecessary experimentation, including the currently considered best mode and preferred embodiments. Nothing in this disclosure should be considered as limiting the scope of the invention, and numerous modifications, equivalents, and substitutions can be made without departing from the scope and spirit of the invention. The scope of the invention should be understood from the appended claims.

[0178] This document describes methods and components. However, similar or equivalent methods and components can also be used to obtain variations of the invention. Materials, articles, components, methods, and embodiments are for illustrative purposes only and are not intended to be limiting.

[0179] Although only a few embodiments have been disclosed in detail above, other embodiments are possible, and the inventors intend that these embodiments be included within the scope of this specification. This specification describes specific examples of achieving a more general objective, which can be achieved in other ways. This disclosure is intended to be exemplary, and the claims are intended to cover any modifications or alternatives that would be foreseeable to those skilled in the art.

[0180] Having illustrated and described the principles of the invention with reference to exemplary embodiments, those skilled in the art will understand that the described examples are illustrative embodiments and modifications may be made to their arrangement and details without departing from these principles. Techniques from any example may be incorporated into one or more other examples. This specification and embodiments are intended to be considered exemplary only, and the true scope and spirit of the invention are indicated by the following claims.

Claims

1. A method of preparing procollagen from freshwater fish, characterized by: The method comprises the following steps (1) removing blood, water and fat from collagen-containing freshwater fish tissue using pressure extrusion; (2) mechanically separating the freshwater fish tissue; (3) mixing the freshwater fish tissue with a surfactant, an electrolyte and urea to obtain an emulsion solution; (4) homogenizing the emulsion solution for 13 to 20 minutes; (5) refrigerating the emulsion solution at 0 to 10°C for 5 to 60 minutes; (6) washing the emulsion solution with water at 38 to 42°C until the absorbance value of the washing liquid at 200 to 300 nm approaches zero to reduce the endotoxin in the freshwater fish tissue to less than 0.25 EU / ml; (7) mixing the freshwater fish tissue with an enzyme and an acid solution with a pH of 3 to 6 to obtain an extract solution; (8) homogenizing the extract solution at 4 to 75°C for 2 to 48 hours; (9) adding an enzyme inhibitor to the extract solution to reduce the pH to less than 3; (10) homogenizing the inactivated extract solution at 4 to 75°C for 2 to 48 hours; (11) separating the supernatant from the homogenized extract solution to obtain the desired procollagen.

2. A method of preparing procollagen from freshwater fish as claimed in claim 1, wherein: The electrolyte is NaCl, and the added amount of NaCl is 1 to 5 wt%.

3. A method of preparing procollagen from freshwater fish as claimed in claim 1, wherein: The added amount of urea is 3 to 10 wt%.

4. A method of preparing procollagen from freshwater fish as claimed in claim 1, wherein: The surfactant is selected from the group consisting of Tween 20, Tween 80, Triton X-100 and mixtures thereof, and the added amount is 0.05 to 0.5 wt%.

5. A method of preparing procollagen from freshwater fish as claimed in claim 1, wherein: The activity of the enzyme is between 20 to 2000 U, and the added amount of the enzyme is 0.5 to 10 wt%.

6. A method of preparing procollagen from freshwater fish as claimed in claim 1, wherein: The enzyme is selected from the group consisting of papain, bromelain and mixtures thereof.

7. A method of preparing procollagen from freshwater fish as claimed in claim 1, wherein: The acid solution is selected from the group consisting of acetic acid, citric acid, lactic acid and mixtures thereof, and the added amount is between 0.5 to 1 M.

8. A method of preparing procollagen from freshwater fish as claimed in claim 6, wherein: The enzyme mixture is a combination of bromelain and papain, and the mixing ratio of bromelain to papain is between 0:1 to 1:

2.

9. A method of preparing procollagen from freshwater fish as claimed in claim 1, wherein: The enzyme inhibitor is selected from the group consisting of acetic acid, lactic acid and mixtures thereof, and the mixing ratio of acetic acid to lactic acid is between 1:0 to 1:

3.

10. A method of preparing procollagen from freshwater fish as claimed in claim 1, wherein: The electrolyte is an organic salt with the form of Na-X.

11. A method of preparing procollagen from freshwater fish as claimed in claim 1, wherein: The mechanical separation of the freshwater fish tissue further comprises cutting and pulverizing the freshwater fish tissue into a form selected from the group consisting of powder, block, strip and combinations thereof.

12. A method of preparing procollagen from freshwater fish as claimed in claim 1, wherein: Separating the supernatant from the homogenized extract solution further comprises a method selected from the group consisting of filtration, centrifugation, screening and combinations thereof.

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