Takifugu fish skin collagen peptide and extraction method thereof

Abstract

The invention discloses a takifugu fish skin collagen peptide and an extraction method thereof, and the extraction method comprises the following steps: adding takifugu fish skin without thorns into a calcareous alkali solution for stirring and washing to remove impure proteins and pigments, filtering and washing with pure water to obtain takifugu fish skin subjected to alkali treatment; adding an acidic extracting solution into the alkali-treated takifugu fish skin, and carrying out hydrothermal reaction under the protection of inert gas, so that the alkali-treated takifugu fish skin is fully dissolved to obtain a takifugu fish skin polypeptide extracting solution; the takifugu fish skin polypeptide extracting solution is filtered, dialyzed and subjected to vacuum freeze drying, and the fish skin collagen peptide is obtained. Under the condition of low energy consumption, the defects that an existing enzymolysis method is high in cost and complex in process and a traditional chemical method seriously damages a polypeptide structure are overcome, and the three-helix structure of the extracted collagen peptide can be effectively protected, so that the collagen peptide can be applied to the fields of cosmetics, functional food and medical materials.

Description

Technical Field

[0001] This invention relates to the field of collagen extraction technology, specifically to a pufferfish skin collagen peptide and its extraction method. Particularly relevant are International Patent Classifications C07K1 / 14 (extraction; separation; purification) and C07K14 / 78 (binding tissue peptides, such as collagen, elastin, laminin, fibronectin, hydrin, cold-insoluble immunoglobulins). Background Technology

[0002] Collagen, also known as collagen protein, is the most abundant and widely distributed glycoprotein in fish skin. Its unique right-handed triple helix structure has attracted widespread attention. Collagen is rich in glycine, proline, and hydroxyproline, and its hydrolysate, collagen peptides, has broad application prospects in the medical, health, and beauty industries. However, collagen is a large-molecule fibrous sclerosing protein with a relative molecular weight as high as 300,000 Daltons (Da). It cannot be directly absorbed by the human body after ingestion; it needs to be digested and broken down into smaller peptides or free amino acids before it can be absorbed and exert its effects. However, the absorption efficiency is very low, only about 2.5%.

[0003] Collagen peptides are produced from fresh animal tissues (skin, bones, tendons, scales, etc.) through extraction, hydrolysis, and refining processes. These products have a relative molecular mass of less than 10,000 Daltons and an absorption efficiency of 90% or higher, exhibiting higher efficacy than pure collagen. Currently, my country's aquaculture and processing industries are developing rapidly, generating large quantities of fish skin, scales, and other byproducts, which are high in collagen. However, current collagen peptide extraction methods primarily focus on common fish such as tilapia and cod, resulting in severe homogenization of raw materials and poor biocompatibility in the prepared peptides. Pufferfish skin, a byproduct of fisheries processing, is abundant in the South my country Sea and rich in protein, but its utilization efficiency is currently low.

[0004] Existing technologies mainly rely on enzymatic hydrolysis, chemical methods, and physical methods for extracting collagen peptides, all of which have significant limitations. Enzymatic hydrolysis is currently the most commonly used method, but enzyme preparations are expensive, subsequent processing costs increase, process conditions are stringent and complex to control, and the purity of the collagen peptides produced is low. Traditional chemical hydrolysis methods (using only strong acids / bases) severely damage the natural spatial structure of amino acids and peptides, leading to the loss or reduction of their biological activities (such as antioxidant, moisturizing, and repairing properties). Physical methods, such as hot water extraction, are inefficient, produce single products, and have limited functional activities.

[0005] Therefore, there is an urgent need to develop a new method for efficiently extracting highly bioactive collagen peptides from pufferfish skin, so as to achieve efficient utilization of marine biological resources. This method aims to overcome the shortcomings of existing enzymatic hydrolysis methods, such as high cost and complex processes, as well as the severe damage to peptide structures caused by traditional chemical methods. The goal is to improve the biocompatibility and bioactivity of collagen peptides, effectively protect the natural structure of the extracted collagen peptides, and thus enable their application in cosmetics, functional foods, and medical materials. Summary of the Invention

[0006] The purpose of this invention is to develop a novel method for efficiently extracting highly bioactive collagen peptides from pufferfish skin, thereby achieving efficient utilization of marine biological resources. This method aims to overcome the drawbacks of existing enzymatic hydrolysis methods (high cost and complex processes) and traditional chemical methods (severe damage to peptide structures). It effectively protects the natural structure of the extracted collagen peptides, enabling their application in cosmetics, functional foods, and medical materials. To solve this technical problem: The first aspect of this patent provides a method for preparing collagen peptides from pufferfish skin, comprising the following steps: Step A: Add the deboned pufferfish skin to a calcium-based alkaline solution and stir to remove impurities, proteins, and pigments. After filtration and washing with pure water, the alkaline-treated pufferfish skin is obtained. Step B: Add acidic extract to the alkali-treated pufferfish skin and carry out a hydrothermal reaction under inert gas protection to completely dissolve the alkali-treated pufferfish skin and obtain pufferfish skin polypeptide extract. Step C: The pufferfish skin polypeptide extract was filtered, dialyzed, and freeze-dried under vacuum to obtain fish skin collagen peptides.

[0007] Furthermore, in step A, the mass-to-volume ratio of the deboned pufferfish skin to the calcium alkali solution is 1:(5~8), 1:(8~15), or 1:(15~20). The unit for the deboned pufferfish skin is g, and the unit for the calcium alkali solution is mL.

[0008] Furthermore, the concentration of the calcium alkaline solution is 3-4%, 4-5%, or 5-7%; the mass percentage concentration of calcium ions in the calcium alkaline solution is 1.6-2.0%, 2.0-2.5%, 2.5-3.0%, or 3.0-3.8%.

[0009] Furthermore, the calcium alkaline solution includes calcium hydroxide suspension, calcium oxide aqueous suspension, or an alkaline aqueous solution composed of a strong base and a soluble calcium salt; wherein the strong base includes sodium hydroxide and / or potassium hydroxide; and the soluble calcium salt includes one or more combinations of calcium chloride, calcium nitrate, calcium acetate, calcium propionate, calcium lactate, and calcium gluconate.

[0010] Furthermore, the agitation speed is 30~80 r / min, 80~100 r / min or 100~150 r / min.

[0011] Furthermore, the washing time is 2~2.5h, 2.5~3.5h, or 3.5~4.0h.

[0012] Furthermore, the pH value of pufferfish skin after alkali treatment is 6.5~7.5.

[0013] Furthermore, in step B, the acidic extraction solution is one or more combinations of citrate-sodium citrate buffer, acetate buffer, or hydrochloric acid solution.

[0014] Furthermore, the acidic extract includes 0.04–0.06 mol / L sodium chloride, and the pH of the acidic extract is 4.0–5.0.

[0015] Furthermore, the mass-to-volume ratio of the alkali-treated pufferfish skin to the acidic extract is 1:(5~8), 1:(8~12), or 1:(12~15).

[0016] Furthermore, the inert gas is one or more combinations of nitrogen, argon, and helium.

[0017] Furthermore, the hydrothermal reaction temperature is 70~75℃, 75~85℃, or 85~90℃; the hydrothermal reaction time is 4~5h, 5~7h, or 7~8h.

[0018] Furthermore, step B does not include enzymatic hydrolysis.

[0019] Furthermore, in step C, the sieve used for filtering is 300-500 mesh.

[0020] Furthermore, the dialysis bag has a retention capacity of 3000-5000 Daltons, 5000-7000 Daltons, or 7000-10000 Daltons.

[0021] Furthermore, the vacuum freeze-drying temperature is -60 to -80°C, and the vacuum freeze-drying time is 24 to 48 hours.

[0022] Another aspect of this patent provides a pufferfish skin collagen peptide, which is prepared by a method for preparing fish skin collagen peptides, and the molecular weight of the fish skin collagen peptides is 3000~5000 Daltons, 5000~7000 Daltons or 7000~10000 Daltons.

[0023] This patent also provides a cosmetic product comprising pufferfish skin collagen peptides prepared by the above-described method for preparing pufferfish skin collagen peptides.

[0024] Furthermore, cosmetics are used for moisturizing or metabolic repair of connective tissue.

[0025] Another aspect of this patent provides a medical dressing comprising pufferfish skin collagen peptides prepared by the above-described method for preparing pufferfish skin collagen peptides.

[0026] Furthermore, medical dressings are used to absorb wound exudate or for wound repair.

[0027] This patent also provides a food product comprising pufferfish skin collagen peptides prepared by the above-described method for preparing pufferfish skin collagen peptides.

[0028] Furthermore, food can be used to supplement calcium, promote intestinal absorption, or promote the metabolic repair of connective tissue.

[0029] Therefore, it can be concluded that the pufferfish skin collagen peptide and its extraction method provided in this application, by subjecting the deboned pufferfish skin to "calcium alkali pretreatment" and "acidic hydrothermal treatment under inert gas protection" combined with vacuum freeze-drying, overcomes the shortcomings of existing enzymatic hydrolysis methods, such as high cost and complex processes, and traditional chemical methods, which severely damage the peptide structure, under low energy consumption conditions. It can effectively protect the natural structure (collagen triple helix structure) of the extracted collagen peptide, thus enabling its application in the fields of cosmetics, functional foods, and medical materials. Attached Figure Description

[0030] The above description of the present invention and the following detailed embodiments will be better understood when read in conjunction with the accompanying drawings. It should be noted that the drawings are merely examples of the claimed technical solutions.

[0031] Figure 1 This is a diagram showing the state of different concentrations of dissolved collagen peptides from pufferfish skin in Test Example 1 of this patent. Figure 2 This is a diagram showing the state of the pufferfish skin collagen peptides after vacuum freeze-drying in Test Example 1 of this patent. Figure 3 The Fourier transform infrared spectrum of Test Example 3 in this patent (where the horizontal axis represents wavenumber, and the unit is (cm)). -1 ); DSP is pufferfish skin collagen peptide; C-FSC is commercial fish skin collagen); Figure 4 The circular dichroism chromatogram of pufferfish skin collagen peptides in Test Example 4 of this patent is shown below (where the horizontal axis represents wavelength in nm and the vertical axis represents circular dichroism in millidograms). Figure 5This is a gel permeation chromatogram of pufferfish skin collagen peptides in Test Example 5 of this patent (where the horizontal axis represents molecular weight in Da; the left vertical axis (dw / dlog M) represents the differential distribution, indicating the mass fraction of molecular weight as a function of the logarithm of molecular weight, corresponding to a bell-shaped curve (peak-shaped curve) composed of black solid dots, with higher peaks indicating more peptide molecules near that molecular weight; the right vertical axis (% Ht) represents the cumulative fractional distribution, indicating the sum of the mass percentages of all components less than or equal to a certain molecular weight, corresponding to an "S"-shaped rising curve composed of red solid dots, where 100% indicates that all molecular weight ranges in the sample have been covered; Mp is the peak molecular weight of 4784 Da, Mn is the number-average molecular weight of 4269 Da, Mw is the weight-average molecular weight of 5498 Da, Mz is the average molecular weight of 7051 Da, MZ+1 is the average molecular weight of all molecules in the polymer chain of 8708 Da, and PDI is the polydispersity index of 1.29). Figure 6 The image shows a scanning electron microscope (SEM) image of pufferfish skin collagen peptides in Test Example 6 of this patent (where A is a 1% pufferfish skin collagen peptide solution; B is a 2% pufferfish skin collagen peptide solution; C is a 4% pufferfish skin collagen peptide solution; and D is an 8% pufferfish skin collagen peptide solution). Detailed Implementation

[0032] The detailed features and advantages of this application are described below in the specific embodiments. The content of this description is sufficient to enable any person skilled in the art to understand the technical content of this application and implement it accordingly. Based on the specification, claims and drawings disclosed in this specification, a person skilled in the art can easily understand the related objectives and advantages of this application.

[0033] In this specification and claims, several terms will be used, and unless otherwise indicated, these terms will be defined to have the following meanings: All other terms used herein for special definition are intended to have the general meaning understood by one of ordinary skill in the art, and in particular, meaning that one of ordinary skill in the art, upon reading the claims, specification and drawings of this patent, can directly and without doubt determine how the technical solution of this patent can be implemented.

[0034] Even if there are incomplete descriptions, omissions, or ambiguities in the grammar, words, punctuation, graphics, symbols, etc. of the claims, specification, and drawings of this patent, a person skilled in the art can still arrive at the only correct understanding by reading the claims, specification, and drawings as a whole without extensive reasoning or experimentation, and effectively exclude various incorrect interpretations that are not aimed at achieving the purpose of this patent.

[0035] The "range" disclosed herein 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 expected that ranges of 60-110 and 80-120 are also expected. 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 "ab" 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.

[0036] Unless otherwise specified, all embodiments and preferred embodiments mentioned herein can be combined to form new technical solutions.

[0037] Unless otherwise specified, all the technical features and preferred features mentioned herein can be combined to form new technical solutions.

[0038] Unless otherwise specified, all steps mentioned herein may be performed sequentially or randomly, but are preferably performed sequentially. For example, the method includes steps (a) and (b), indicating that the method may include steps (a) and (b) performed sequentially, or it may include steps (b) and (a) performed sequentially. For example, the mention that the method may also include step (c) indicates that step (c) may be added to the method in any order; for example, the method may include steps (a), (b), and (c), or it may include steps (a), (c), and (b), or it may include steps (c), (a), and (b), etc.

[0039] Unless otherwise specified, the terms "comprising" and "including" as used herein can be open-ended or closed-ended. For example, "comprising" and "including" may mean that other components not listed may also be included, or that only the listed components may be included.

[0040] In the description of this article, it should be noted that, unless otherwise stated, "above" and "below" include the number itself, and "several" in "one or more" means two or more.

[0041] In this description, unless otherwise stated, the term "or" is inclusive. For example, the phrase "A or B" means "A, B, or both A and B". More specifically, the condition "A or B" is satisfied by any of the following conditions: A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); or both A and B are true (or exist).

[0042] Unless otherwise specified, percentages (%) in this document refer to percentages by mass relative to the composition.

[0043] Unless otherwise stated herein, the sum of the contents of the components in the composition is 100%.

[0044] In this document, unless otherwise stated, “combination of” means a multi-component mixture of the elements, such as two, three, four, and up to the maximum possible multi-component mixture.

[0045] Unless otherwise specified, the term "a" as used in this specification means "at least one".

[0046] The terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise explicitly specified.

[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0048] To make the objectives, technical solutions, and advantages of the present invention clearer, the embodiments of the present invention will be further described in detail below with reference to the accompanying drawings. Unless otherwise specified, the experimental methods described in the embodiments of the present invention are conventional methods, and the materials and reagents used in the following embodiments are commercially available unless otherwise specified.

[0049] (1) Source of sample materials Six-spotted pufferfish: caught in Qinglan Port, Wenchang City, Hainan Province.

[0050] (2) Sources of reagents and consumables Table 1. Sources of reagents and consumables required for the experiment ;

[0051] (3) Source of instruments and equipment Table 2. Sources and Models of Instruments and Equipment Required for the Experiment ;

[0052] Example 1 A pufferfish skin collagen peptide and its extraction method, comprising the following steps: S1. Pre-treatment of pufferfish skin, the specific steps are as follows: (1) Anesthetize fresh pufferfish, peel off the pufferfish skin under anesthesia in a clean bench, and soak the pufferfish skin in 4℃ deionized water for 8 hours to clean and remove surface impurities, thus obtaining clean pufferfish skin.

[0053] (2) Cut the clean pufferfish skin obtained in step (1) into pieces and place it in an ultrasonic cleaner for ultrasonic cavitation. Continue ultrasonication for 20 minutes under an ultrasonic power of 200 W to obtain pretreated pufferfish skin.

[0054] S2. Alkali treatment, the specific steps are as follows: (1) Add a 3% calcium hydroxide suspension to the pretreated pufferfish skin obtained in step S1 above. The mass-volume ratio of the pretreated pufferfish skin (unit: g) to the 3% calcium hydroxide suspension (unit: mL) is 1:5 to obtain the first mixture. In some other specific embodiments, other calcium-based alkaline solutions can also be used to treat the pretreated pufferfish skin with alkali. These other calcium-based alkaline solutions include calcium hydroxide suspension, calcium oxide aqueous suspension, or alkaline solutions composed of a strong base and a soluble calcium salt. The strong base is selected from one or a combination of sodium hydroxide and potassium hydroxide. The soluble calcium salt includes one or a combination of calcium chloride, calcium nitrate, calcium acetate, calcium propionate, calcium lactate, and calcium gluconate. The pH value of the other calcium-based alkaline solutions is 11.0 to 13.0, and the mass percentage concentration of calcium ions in the other calcium-based alkaline solutions is 1.6% to 3.8%.

[0055] (2) The first mixture obtained in step (1) was stirred for the first time using a magnetic stirrer (commercially available). The stirring speed for the first time was 30 r / min, and the stirring time was 4 h, so that the calcium hydroxide suspension could fully wash the pretreated pufferfish skin to remove the impurities and pigments on the pretreated pufferfish skin. At the same time, if the alkalinity is too weak (pH value less than 11.0), it will not be able to remove the impurities and pigments on the pufferfish skin, and if the alkalinity is too strong (pH value greater than 13.0), it will seriously damage the natural spatial structure of amino acids and peptides.

[0056] (3) The pretreated pufferfish skin that has been removed from impurities and pigments in step (2) above is repeatedly washed with deionized water so that the pH value of the pretreated pufferfish skin that has been removed from impurities and pigments is neutral (neutral is defined as pH=6.5~7.5) to obtain alkali-treated pufferfish skin.

[0057] S3. Acid treatment, the specific steps are as follows: (1) Weigh 19.21 g of anhydrous citric acid (molar mass of 192.12 g / mol) and dissolve it in 800 mL of deionized water. Add sodium citrate to adjust the pH to 4.5, then add 2.92 g of sodium chloride and stir thoroughly to dissolve. Then use deionized water to make up to 1 L to prepare a citric acid-sodium citrate buffer solution with a concentration of 0.1 mol / L, so that the final concentration of sodium chloride in the buffer solution system is 0.05 mol / L. In some other specific embodiments, a citrate-sodium citrate buffer solution with a pH of 4.0-5.0 and a concentration of 0.05-0.20 mol / L can also be prepared according to the above method, so that the final concentration of sodium chloride in the buffer solution system is 0.04-0.06 mol / L.

[0058] (2) Add 0.1 mol / L citrate-sodium citrate buffer to the alkali-treated pufferfish skin obtained in step S2 above. The mass-volume ratio of the alkali-treated pufferfish skin (unit: g) to the citrate-sodium citrate buffer (unit: mL) is 1:5 to obtain a second mixture. In some other specific embodiments, an acetate-sodium acetate buffer solution with a pH of 4.0-5.0 and a concentration of 0.1-0.5 mol / L can also be prepared, or a hydrochloric acid buffer solution with a pH of 1.0-3.0 and a concentration of 0.1-0.5 mol / L can be prepared, each containing sodium chloride at a final concentration of 0.04-0.06 mol / L, and then mixed with the alkali-treated pufferfish skin, which has the same effect as the citrate-sodium citrate buffer solution.

[0059] (3) The second mixture obtained in step (2) is stirred a second time using a magnetic stirrer at a speed of 50 r / min for 4 h. Under the protective condition of nitrogen as the inert gas, a hydrothermal reaction is carried out in a reactor at a temperature of 70°C for 8 h to completely dissolve the alkali-treated pufferfish skin and form a slightly yellow transparent liquid, thus obtaining the pufferfish skin polypeptide extract. In some other specific embodiments, the inert gas can also be argon or helium, which has the same effect as nitrogen. At the same time, excessively high or low hydrothermal reaction temperature, as well as excessively short or long hydrothermal time, will lead to poor acid treatment results.

[0060] S4. Post-processing, the specific steps are as follows: (1) Place the pufferfish skin polypeptide extract obtained in step S3 above in an ice-water bath to rapidly lower the temperature of the pufferfish skin polypeptide extract to below 15°C, and then filter it using a 300-mesh sieve to obtain the filtrate. In some other specific embodiments, a 300-500 mesh sieve can also be used for filtration to obtain the filtrate.

[0061] (2) Place the filtrate obtained in step (1) above into a dialysis bag and dialyze it in flowing deionized water for 24 h to remove citrate and sodium chloride, so that the molecular weight of the filtrate in the dialysis bag is maintained in the range of 5000 Daltons (Da), and the dialysate is obtained. In some other specific embodiments, the molecular weight of the dialyzed solution can also be maintained in the range of 5000~10000 Da.

[0062] (3) The dialysate obtained in step (2) above is placed in a freeze dryer for vacuum freeze drying. The temperature of vacuum freeze drying is -60℃, the vacuum degree is less than 10 Pa, and the time of vacuum freeze drying is 48 h to obtain pufferfish skin collagen peptides.

[0063] Example 2 S1. Pre-treatment of pufferfish skin, the specific steps are as follows: (1) Anesthetize fresh pufferfish, peel off the pufferfish skin under anesthesia in a clean bench, and soak the pufferfish skin in 4℃ deionized water for 12 h to clean and remove surface impurities to obtain clean pufferfish skin.

[0064] (2) Cut the clean pufferfish skin obtained in step (1) into pieces and place it in an ultrasonic cleaner for ultrasonic cavitation. Under the condition of ultrasonic power of 300 W, continue ultrasonication for 10 min to obtain pretreated pufferfish skin.

[0065] S2. Alkali treatment, the specific steps are as follows: (1) Add a 7% calcium hydroxide suspension to the pretreated pufferfish skin obtained in step S1 above. The mass-volume ratio of the pretreated pufferfish skin to the 7% calcium hydroxide suspension is 1:20 to obtain the first mixture. In some other specific embodiments, other calcium-based alkaline solutions can also be used to treat the pretreated pufferfish skin with alkali. These other calcium-based alkaline solutions include calcium hydroxide suspension, calcium oxide aqueous suspension, or alkaline solutions composed of a strong base and a soluble calcium salt. The strong base is selected from one or a combination of sodium hydroxide and potassium hydroxide. The soluble calcium salt includes one or a combination of calcium chloride, calcium nitrate, calcium acetate, calcium propionate, calcium lactate, and calcium gluconate. The pH value of the other calcium-based alkaline solutions is 11.0 to 13.0, and the mass percentage concentration of calcium ions in the other calcium-based alkaline solutions is 1.6% to 3.8%.

[0066] (2) The first mixture obtained in step (1) above is stirred for the first time using a magnetic stirrer (commercially available). The stirring speed for the first time is 150 r / min, and the stirring time is 2 h, so that the calcium hydroxide suspension can fully wash the pretreated pufferfish skin to remove the impurities and pigments on the pretreated pufferfish skin. At the same time, if the alkalinity is too weak, it will not be able to remove the impurities and pigments on the pufferfish skin, and if the alkalinity is too strong, it will seriously damage the natural spatial structure of amino acids and peptides.

[0067] (3) The pretreated pufferfish skin that has been removed from impurities and pigments in step (2) above is repeatedly washed with deionized water so that the pH value of the pretreated pufferfish skin that has been removed from impurities and pigments is neutral (neutral is defined as pH=6.5~7.5) to obtain alkali-treated pufferfish skin.

[0068] S3. Acid treatment, the specific steps are as follows: (1) Weigh 19.21 g of anhydrous citric acid (molar mass of 192.12 g / mol) and dissolve it in 800 mL of deionized water. Add sodium citrate to adjust the pH to 4.5, then add 2.92 g of sodium chloride and stir thoroughly to dissolve. Then use deionized water to make up to 1 L to prepare a citric acid-sodium citrate buffer solution with a concentration of 0.1 mol / L, so that the final concentration of sodium chloride in the buffer solution system is 0.05 mol / L. In some other specific embodiments, a citrate-sodium citrate buffer solution with a pH of 4.0-5.0 and a concentration of 0.05-0.20 mol / L can also be prepared according to the above method, so that the final concentration of sodium chloride in the buffer solution system is 0.04-0.06 mol / L.

[0069] (2) Add 0.1 mol / L citrate-sodium citrate buffer to the alkali-treated pufferfish skin obtained in step S2 above. The mass-volume ratio of alkali-treated pufferfish skin (unit: g) to citrate-sodium citrate buffer (unit: mL) is 1:15 to obtain a second mixture. In some other specific embodiments, an acetate-sodium acetate buffer solution with a pH of 4.0-5.0 and a concentration of 0.1-0.5 mol / L can also be prepared, or a hydrochloric acid buffer solution with a pH of 1.0-3.0 and a concentration of 0.1-0.5 mol / L can be prepared, each containing sodium chloride at a final concentration of 0.04-0.06 mol / L, and then mixed with the alkali-treated pufferfish skin, which has the same effect as the citrate-sodium citrate buffer solution.

[0070] (3) The second mixture obtained in step (2) is stirred a second time using a magnetic stirrer at a speed of 150 r / min for 2 h. Under the protective condition of argon as the inert gas, a hydrothermal reaction is carried out in a reactor at a temperature of 90°C for 4 h to completely dissolve the alkali-treated pufferfish skin and form a slightly yellow transparent liquid, thus obtaining the pufferfish skin polypeptide extract. In some other specific embodiments, the inert gas can also be nitrogen or helium, which has the same effect as argon. At the same time, excessively high or low hydrothermal reaction temperature, as well as excessively short or long hydrothermal time, will lead to poor acid treatment results.

[0071] S4. Post-processing, the specific steps are as follows: (1) Place the pufferfish skin polypeptide extract obtained in step S3 above in an ice-water bath to rapidly lower the temperature of the pufferfish skin polypeptide extract to below 15°C, and then filter it using a 500-mesh sieve to obtain the filtrate. In some other specific embodiments, a 300-500 mesh sieve can also be used for filtration to obtain the filtrate.

[0072] (2) Place the filtrate obtained in step (1) above into a dialysis bag and dialyze it in flowing deionized water for 24 h to remove citrate and sodium chloride, so that the molecular weight of the filtrate in the dialysis bag is maintained in the range of 5000 Daltons (Da), and the dialysate is obtained. In some other specific embodiments, the molecular weight of the dialyzed solution can also be maintained in the range of 5000~10000 Da.

[0073] (3) The dialysate obtained in step (2) above is placed in a freeze dryer for vacuum freeze drying. The temperature of vacuum freeze drying is -80℃, the vacuum degree is less than 10 Pa, and the time of vacuum freeze drying is 24 h to obtain pufferfish skin collagen peptides.

[0074] Example 3 S1. Pre-treatment of pufferfish skin, the specific steps are as follows: (1) Weigh 1.0 kg of commercially available dried pufferfish skin, remove the fish bones by hand or machine, and cut it into 2 cm × 2 cm pieces.

[0075] (2) Soak the skin pieces obtained in step (1) in deionized water for 4 to 12 hours to thoroughly soak the dried pufferfish skin until it is soft. Clean the surface impurities to obtain clean pufferfish skin.

[0076] (3) Cut the clean pufferfish skin obtained in step (2) into pieces and place it in an ultrasonic cleaner for ultrasonic cavitation. Continue ultrasonication for 10 minutes under an ultrasonic power of 300 W to obtain pretreated pufferfish skin.

[0077] S2. Alkali treatment, the specific steps are as follows: (1) Add a 4% calcium oxide aqueous suspension to the pretreated pufferfish skin obtained in step S1 above. The mass-volume ratio of the pretreated pufferfish skin to the 4% calcium oxide aqueous suspension is 1:10, that is, add 10 L of the 4% calcium oxide aqueous suspension and mix to obtain the first mixture. In some other specific embodiments, other calcium-based alkaline solutions can also be used to treat the pretreated pufferfish skin with alkali. These other calcium-based alkaline solutions include calcium hydroxide suspensions or alkaline solutions composed of a strong base and a soluble calcium salt. The strong base is selected from one or a combination of sodium hydroxide and potassium hydroxide. The soluble calcium salt includes one or a combination of calcium chloride, calcium nitrate, calcium acetate, calcium propionate, calcium lactate, and calcium gluconate. The pH value of the other calcium-based alkaline solutions is 11.0 to 13.0, and the mass percentage concentration of calcium ions in the other calcium-based alkaline solutions is 1.6% to 3.8%.

[0078] (2) The first mixture obtained in step (1) above is stirred for the first time using a magnetic stirrer (commercially available). The stirring speed for the first time is 80 r / min, and the stirring time is 3 h, so that the calcium hydroxide suspension can fully wash the pretreated pufferfish skin to remove the impurities and pigments on the pretreated pufferfish skin. At the same time, if the alkalinity is too weak, it will not be able to remove the impurities and pigments on the pufferfish skin, and if the alkalinity is too strong, it will seriously damage the natural spatial structure of amino acids and peptides.

[0079] (3) The pretreated pufferfish skin that has been removed from impurities and pigments in step (2) above is repeatedly washed with deionized water so that the pH value of the pretreated pufferfish skin that has been removed from impurities and pigments is neutral (neutral is defined as pH=6.5~7.5) to obtain alkali-treated pufferfish skin.

[0080] S3. Acid treatment, the specific steps are as follows: (1) Weigh 19.21 g of anhydrous citric acid (molar mass of 192.12 g / mol) and dissolve it in 800 mL of deionized water. Add sodium citrate to adjust the pH to 4.5, then add 2.92 g of sodium chloride and stir thoroughly to dissolve. Then use deionized water to make up to 1 L to prepare a citric acid-sodium citrate buffer solution with a concentration of 0.1 mol / L, so that the final concentration of sodium chloride in the buffer solution system is 0.05 mol / L. In some other specific embodiments, a citrate-sodium citrate buffer solution with a pH of 4.0-5.0 and a concentration of 0.05-0.20 mol / L can also be prepared according to the above method, so that the final concentration of sodium chloride in the buffer solution system is 0.04-0.06 mol / L.

[0081] (2) Add 0.1 mol / L citrate-sodium citrate buffer to the alkali-treated pufferfish skin obtained in step S2 above. The mass-volume ratio of alkali-treated pufferfish skin (unit: g) to citrate-sodium citrate buffer (unit: mL) is 1:15 to obtain a second mixture. In some other specific embodiments, an acetate-sodium acetate buffer solution with a pH of 4.0-5.0 and a concentration of 0.1-0.5 mol / L can also be prepared, or a hydrochloric acid buffer solution with a pH of 1.0-3.0 and a concentration of 0.1-0.5 mol / L can be prepared, each containing sodium chloride at a final concentration of 0.04-0.06 mol / L, and then mixed with the alkali-treated pufferfish skin, which has the same effect as the citrate-sodium citrate buffer solution.

[0082] (3) The second mixture obtained in step (2) is stirred a second time using a magnetic stirrer at a speed of 100 r / min for 4 h. A hydrothermal reaction is then carried out under nitrogen atmosphere at a temperature of 80°C for 6 h to completely dissolve the alkali-treated pufferfish skin, forming a slightly yellow, transparent liquid, thus obtaining the pufferfish skin polypeptide extract. In some other embodiments, the inert gas can also be argon or helium, which has the same effect as nitrogen. Furthermore, excessively high or low hydrothermal reaction temperatures, as well as excessively short or long hydrothermal times, will result in poor acid treatment results.

[0083] S4. Post-processing, the specific steps are as follows: (1) Place the pufferfish skin polypeptide extract obtained in step S3 above in an ice water bath to rapidly reduce the temperature of the pufferfish skin polypeptide extract to below 15°C, and then filter it using a 500-mesh sieve to obtain the filtrate; in some other specific embodiments, a 300-500-mesh sieve can also be used for filtration to obtain the filtrate.

[0084] (2) Place the filtrate obtained in step (1) above into a dialysis bag and dialyze it in flowing deionized water for 24 h to remove citrate and sodium chloride, so that the molecular weight of the filtrate in the dialysis bag is maintained in the range of 5000 Daltons (Da) to obtain dialysate; in some other specific embodiments, the molecular weight of the dialyzed filtrate can also be maintained in the range of 5000~10000 Da.

[0085] (3) The dialysate obtained in step (2) above is placed in a freeze dryer for vacuum freeze drying. The temperature of vacuum freeze drying is -70℃, the vacuum degree is less than 10 Pa, and the time of vacuum freeze drying is 36 h to obtain pufferfish skin collagen peptides.

[0086] Comparative Example Compared with Example 1, in this comparative example, after obtaining alkali-treated pufferfish skin, collagen peptides from the pufferfish skin were prepared using a conventional enzymatic hydrolysis method. The specific method is as follows: Alkali-treated pufferfish skin (g) and deionized water (mL) were mixed at a mass-to-volume ratio of 1:10. The mixture was heated to 50°C and then 2% (by mass) of alkaline protease (Alcalase) was added for isothermal enzymatic hydrolysis for 6 h. The mixture was then heated to 90°C for 10 min to inactivate the enzyme. After centrifugation, the supernatant was retained and then filtered, dialyzed (5000 Da), and freeze-dried under vacuum to obtain pufferfish skin collagen peptides.

[0087] Test Example 1: Statistical Analysis of Collagen Peptide Extraction Rate from Pufferfish Skin The extraction rates of pufferfish skin collagen peptides extracted in Examples 1-3 and the comparative examples were statistically analyzed, and the extraction rate calculation formula is shown below: Extraction rate (%) = M1 / M0; Where M1 is the weight of pufferfish skin collagen peptides in g; M0 is the weight of cleaned pufferfish skin in g.

[0088] The results showed that the extraction rate of pufferfish skin collagen peptides obtained by the extraction methods in Examples 1-3 was 27.52%, and there was no significant difference in the extraction rate obtained by the extraction methods in Examples 1-3.

[0089] like Figure 1As shown, the pufferfish skin collagen peptides obtained using the extraction method in Example 1 are as follows: Before vacuum freeze-drying, the pufferfish skin collagen peptides are a transparent liquid at room temperature; the higher the concentration, the thicker the solution and the darker the color. At temperatures below 4°C, the pufferfish skin collagen peptides exhibit a hydrogel-like state. Figure 2 As shown, after vacuum freeze-drying, the pufferfish skin collagen peptides appear as white powdery crystals with high water solubility. They are soluble in water at room temperature, and the higher the temperature, the faster the dissolution rate. After dissolution, they form a clear and colorless solution.

[0090] In contrast, although the extraction rate of pufferfish skin collagen peptides prepared by the conventional enzymatic hydrolysis method in the comparative example was 80.4%, which was significantly higher than the extraction rate of pufferfish skin collagen peptides in the examples of this application, after vacuum freeze-drying, the pufferfish skin collagen peptides in the comparative example appeared as a slightly yellow powder with a fishy smell, and the solution was slightly turbid after dissolution.

[0091] Test Example 2: Amino acid composition analysis of pufferfish skin collagen peptides The amino acid composition analysis of the pufferfish skin collagen peptides extracted in Examples 1-3 and the comparative example is as follows: (1) Accurately weigh 10 mg of pufferfish skin collagen peptides and place them in a reaction flask. Add 1 mL of 6 M hydrochloric acid solution and perform acid hydrolysis at 110 °C for 24 h. (2) Weigh 0.2 mL of the acid-hydrolyzed liquid from step (1) above, add 535 μL of 2 M sodium hydroxide solution for neutralization, and vortex mix well; (3) Using AccQ The AccQ reagent in the Tag Ultra “3X” Derivatization Kit (all reagents below are derived from this kit) Tag Ultra Borate buffer is used to dilute the neutralized and mixed liquid in step (2) above to a 10-fold dilution to obtain the sample to be tested; (4) Weigh 10 μL of the sample to be tested in step (3) into the reaction flask, and add 70 μL of AccQ. TagUltraBorate buffer and 20 μL AccQ Tag reagent was vortexed and heated at 55°C for 10 min. After cooling to room temperature, data were acquired using ultra-high performance liquid chromatography and high resolution mass spectrometry, and the acquired data were analyzed.

[0092] Since Examples 2 and 3 have similar results to Example 1, the amino acid composition of the pufferfish skin collagen peptide extracted in Example 1 was analyzed.

[0093] The results are shown in Table 3. The amino acid composition of the pufferfish skin collagen peptides in Example 1 exhibits typical collagen characteristics. (1) The glycine content is as high as 16.4%, which conforms to the Gly-XY repeat sequence pattern; (2) The total content of imino acids reached 26.4%, including 18.5% proline and 7.9% hydroxyproline. The presence of hydroxyproline directly confirms the collagen properties of the pufferfish skin collagen peptides, and the ratio of proline to hydroxyproline is about 2.4, which can preliminarily prove the integrity of the triple helix structure. (3) The total content of acidic amino acids (total content 26.52%: including 17.95% glutamic acid and 8.57% aspartic acid) was significantly higher than that of basic amino acids (total content 26.52%: including 10.32% arginine and 2.74% lysine), which gave the collagen peptides of pufferfish skin stronger hydrophilicity, and the high arginine content could significantly enhance cell adhesion activity.

[0094] In summary, these characteristics give pufferfish skin collagen peptides significant advantages in moisturizing and promoting wound repair, making them more suitable for cosmetics and biomedical materials.

[0095] Table 3. Statistical analysis results of amino acid composition of pufferfish skin collagen peptides in Example 1 ;

[0096] In contrast, as shown in Table 4, the glycine (Gly) and hydroxyproline (Hypro) contents in the comparative examples were significantly lower than those in Example 1. This demonstrates that while the enzymatic hydrolysis method achieves a high extraction rate of 80.4%, it introduces a large amount of non-collagen components. The total imino acid (Pro+Hypro) content in Example 1 is as high as 26.38%, while it is only about 19.9% ​​in the comparative examples; this explains at the molecular level why the enzymatic product cannot maintain the triple helix structure (leading to the disappearance of the positive peak at 220 nm in the CD spectrum). At the same time, the proportions of leucine (Leu), isoleucine (Ile), and phenylalanine (Phe) in the comparative examples are significantly higher than those in Example 1, further confirming that the present invention achieves "refining" of collagen components through the "calcium alkali pretreatment + nitrogen-protected hydrothermal" process. Although the extraction rate of pufferfish skin collagen peptides in this application is only 27.52%, the purity and bioactivity of pufferfish skin collagen peptides far exceed those of conventional enzymatic hydrolysis products.

[0097] Table 4. Statistical analysis results of amino acid composition of pufferfish skin collagen peptides in the comparative examples. ;

[0098] Test Example 3: Fourier Transform Infrared Spectroscopy of Collagen Peptides from Pufferfish Skin The pufferfish skin collagen peptides prepared in Examples 1-3 and the comparative examples were tested using Fourier transform infrared spectroscopy.

[0099] Weigh 1 mg each of the pufferfish skin collagen peptide (DSP) prepared in Examples 1-3 and the comparative example, add 100 mg of dry potassium bromide powder, mix and place in an agate mortar for grinding. During grinding, the sample and the agate mortar are placed under a drying lamp to ensure dryness, thus obtaining the sample to be tested. Then, spread the sample evenly in a tableting mold, compress it into a tablet, and measure it using a Fourier transform infrared spectrometer with a spectral resolution of 2 cm⁻¹. -1 The measurement range is 400~4000 cm. -1 The number of scans was 30, with commercial fish-skin collagen (C-FSC) used as a control.

[0100] Since the results of Examples 2-3 are similar to those of Example 1, the results will be explained using the pufferfish skin collagen peptide prepared in Example 1 as an example.

[0101] The results are as follows Figure 3 As shown, the two groups of pufferfish skin collagen peptides (labeled DSP) and commercial fish skin collagen (labeled C-FSC) were at 3332.39 cm⁻¹. -1 and 3361.61 cm -1 A broad and strong NH stretching vibration peak appears at 2947.88 cm⁻¹. -1 and 2956.33 cm -1 Asymmetric stretching vibrations of -CH2 appear at 1657.04 cm⁻¹. -1 and 1650.70 cm -1 The C=O stretching vibration of amide I is present at a point, particularly at 1657.04 cm⁻¹. -1 The amide I band at 1542.95 cm⁻¹ exhibits a clear and sharp characteristic peak, indicating that the primary structure and backbone structure of the polypeptide chain are well preserved; -1 and 1540.84 cm -1 The NH bending vibration of amide II appears at 1236.61 cm⁻¹. -1 and 1242.95 cm -1 An NH deformation peak of amide III appeared at the position. Therefore, the absorption peak position of the pufferfish skin collagen peptide prepared in Example 1 is highly consistent with that of commercial fish skin collagen, and it has the same chemical properties as collagen.

[0102] In contrast, the collagen peptides from pufferfish skin in the comparative group (enzymatic hydrolysis group) were at 1650 cm⁻¹-1 The peak shape of amide I in the vicinity shows a significant broadening and red shift, while the peak shape of amide III (1236.61 cm⁻¹) shows a significant red shift. -1 The peak intensity (near the [location]) decreased significantly. This indicates that conventional enzymatic hydrolysis processes severely damage the secondary structure of pufferfish skin collagen peptides. Extensive enzymatic cleavage degrades the fish skin tissue into disordered peptide segments, severely damaging the intramolecular hydrogen bond network. While this significantly increases the extraction rate of pufferfish skin collagen peptides, it results in the loss of the unique bioactive structure of collagen. Furthermore, the forced introduction of other proteins leads to poor product color and odor. In contrast, this application utilizes a calcium alkali pretreatment combined with a gentle hydrothermal reaction under nitrogen protection, exhibiting strong structural recognition and minimizing damage to collagen-specific functional groups.

[0103] Test Example 4: Circular Dichroism Spectrometry Determination of Collagen Peptides from Pufferfish Skin The secondary structures of the pufferfish skin collagen peptides prepared in Examples 1-3 and the comparative examples were tested using a circular dichroism chromatograph.

[0104] Collagen peptides from pufferfish skin were dissolved in deionized water to obtain a peptide solution with a concentration of 0.25 mg / mL. The solution was then placed in a 1 mm quartz cuvette and detected using a circular dichroism spectrometer in the wavelength range of 190 nm to 260 nm, with a step size of 1 nm and a scan rate of 120 nm / min. Type I collagen was used as a blank control. The experiment was performed three times, and the average value was used to plot a curve, which was then smoothed.

[0105] The results are as follows Figure 4 As shown, since Examples 2-3 have similar results to Example 1, the results are illustrated using the pufferfish skin collagen peptide prepared in Example 1 as an example. The pufferfish skin collagen peptide in Example 1 exhibits a strong negative absorption peak at 196-198 nm and a clear weak positive absorption peak near 221 nm. This spectral characteristic is a typical marker of the presence of the triple helix structure of collagen.

[0106] In contrast, while the pufferfish skin collagen peptides in the comparative example exhibited a negative peak near 197 nm, their positive peak near 221 nm essentially disappeared, and the curve became flat. The conventional enzymatic digestion method used in the comparative example resulted in the random and violent cleavage of the pufferfish skin collagen peptide chains, and its spatial conformation was completely transformed into a random coil structure.

[0107] The pufferfish skin collagen peptides prepared in this application, while degrading into small molecule peptides (Mw approximately 5498 Da), successfully retained their biologically active triple helix conformation. Therefore, further mixing the pufferfish skin collagen peptides prepared in Examples 1-3 with commercially available cosmetic glycerin can produce cosmetics, or mixing them with commercially available medical materials can prepare medical dressings. These combinations provide a more robust molecular basis for UV damage repair, moisturizing, skin tissue regeneration, or wound repair, exhibiting significantly superior biological performance compared to collagen products obtained through conventional enzymatic hydrolysis. Alternatively, combining the pufferfish skin collagen peptides prepared in Examples 1-3 with commercially available functional foods can supplement calcium, promote intestinal absorption, or promote the metabolic repair of connective tissue.

[0108] Test Example 5: Gel Permeation Chromatography Test of Collagen Peptides from Pufferfish Skin Gel permeation chromatography (GPC) achieves molecular weight determination based on differences in molecular hydrodynamics. Because substances of different molecular weights have different pathways and require different times to pass through a gel column, molecules of different molecular weights can be separated and quantitatively characterized at high resolution.

[0109] The pufferfish skin collagen peptides prepared in Examples 1-3 were subjected to gel permeation chromatography. Since the results of Examples 2-3 are similar to those of Example 1, the results of the pufferfish skin collagen peptides prepared in Example 1 will be used as an example for explanation.

[0110] The results are as follows Figure 5 As shown, the pufferfish skin collagen peptides prepared in Example 1 of this application exhibit a typical unimodal distribution. The molecular weight of the pufferfish skin collagen peptides is between 1 and 22 kDa, with an average molecular weight (Mz) of 7051 Da, a weight-average molecular weight (Mw) of 5498 Da, a peak molecular weight (Mp) of 4784 Da, a number-average molecular weight (Mn) of 4269 Da, an average molecular weight (MZ+1) of 8708 Da, and a polydispersity index (PDI) of 1.29 > 1. The results indicate that the pufferfish skin collagen peptides have a narrow molecular weight distribution, which allows for precise control of the degradation degree of pufferfish skin collagen. The product components are uniform, and the molecular weight distribution range is concentrated, close to a monodisperse system. It is a safe biomaterial with high uniformity. At the same time, the differential curve (dw / dlog M) has no obvious tailing in the low molecular weight region, which proves that the acidic hydrothermal reaction under inert gas protection effectively reduces the excessive degradation of pufferfish skin collagen peptides.

[0111] Test Example 6: Scanning Electron Microscopy Observation of Collagen Peptides from Pufferfish Skin The internal structure of the pufferfish skin collagen peptides prepared in Examples 1-3 was further observed using a scanning electron microscope.

[0112] The results are as follows Figure 6 As shown, pufferfish skin collagen peptides of different concentrations all exhibit a network-like internal structure. With increasing concentration, the degree of intermolecular bonding increases, and the internal network structure changes from loose to dense, with the mesh size decreasing. However, the 1% and 2% concentrations of pufferfish skin collagen peptides, due to their insufficient mechanical strength to support the internal structure, exhibited pore fragmentation.

[0113] Therefore, it can be concluded that the pufferfish skin collagen peptide and its extraction method provided in this application, by subjecting the deboned pufferfish skin to "calcium alkali pretreatment" and "acidic hydrothermal treatment under inert gas protection" combined with vacuum freeze-drying, overcomes the shortcomings of existing enzymatic hydrolysis methods, such as high cost and complex processes, and traditional chemical methods, which severely damage the peptide structure, under low energy consumption conditions. It can effectively protect the natural structure (collagen triple helix structure) of the extracted collagen peptide, thus enabling its application in the fields of cosmetics, functional foods, and medical materials.

[0114] In the foregoing description of exemplary embodiments / specific implementations of this patent, various features of this patent are sometimes combined in a single embodiment / specific implementation or its figures and description, with the aim of simplifying the disclosure and aiding in the understanding of one or more of the various aspects of the invention. However, unless expressly stated otherwise or in obvious technical contradiction or exclusion, the descriptive method of this patent should not be construed as reflecting an intention that the claimed features of the invention are more than expressly stated in each claim. Rather, the inventive aspect reflected in the claims lies in not all the features of a single foregoing disclosed embodiment / specific implementation. Therefore, the claims following the detailed description are hereby expressly incorporated into this detailed description, each claim existing independently as a separate embodiment / specific implementation of this patent.

[0115] The terms and expressions used in this specification are for illustrative purposes and not for limitation. Their use is not intended to exclude any equivalents of the shown and described features or portions thereof, but rather to facilitate the understanding that various modifications may be possible within the scope of this patent claim. Therefore, it should be understood that while this patent has been specifically disclosed through preferred embodiments, exemplary embodiments, and optional features, variations or modifications of the concepts disclosed herein may be adopted by those skilled in the art, and such variations and modifications are therefore considered to be within the scope of this patent as defined by the appended claims. The specific embodiments given in this specification are examples of useful embodiments of this patent, and it will be apparent to those skilled in the art that this patent can be implemented using many variations of the devices, device components, and method steps disclosed herein.

[0116] The foregoing description of specific embodiments fully discloses the general features of this patent, enabling others to easily modify and / or adapt such embodiments for various applications by applying knowledge within the scope of the art, without excessive experimentation or deviation from the general concept of this patent. Therefore, based on the teachings and guidance provided herein, it is intended that such modifications and alterations be included within the meaning and scope of equivalents of the disclosed embodiments. It should be understood that the wording or terminology used herein is for descriptive purposes and not intended to be limiting; thus, the wording or terminology in this specification will be interpreted by those skilled in the art based on the foregoing teachings and guidance.

[0117] Furthermore, the scope of this patent should not be limited to any of the exemplary embodiments described above, but only to the appended claims and their equivalents.

Claims

1. A method for preparing collagen peptides from pufferfish skin, characterized in that, Includes the following steps: Step A: Add the deboned pufferfish skin to a calcium-alkali solution and stir to remove impurities, proteins, and pigments. After filtration and washing with pure water, the alkali-treated pufferfish skin is obtained. The mass-to-volume ratio of the deboned pufferfish skin to the calcium-alkali solution is 1:(5~8), 1:(8~15), or 1:(15~20). The concentration of the calcium-alkali solution is 3~4%, 4~5%, or 5~7%. The mass percentage concentration of calcium ions in the calcium-alkali solution is 1.6~2.0%, 2.0~2.5%, 2.5~3.0%, or 3.0~3.8%. Step B: Add acidic extract to the alkali-treated pufferfish skin and carry out a hydrothermal reaction under inert gas protection to fully dissolve the alkali-treated pufferfish skin and obtain pufferfish skin polypeptide extract. Step C: The pufferfish skin polypeptide extract is filtered, dialyzed, and freeze-dried under vacuum to obtain the fish skin collagen peptide.

2. The method for preparing pufferfish skin collagen peptides according to claim 1, characterized in that, In step A The calcium alkaline solution includes calcium hydroxide suspension, calcium oxide aqueous suspension, or an alkaline aqueous solution composed of a strong base and a soluble calcium salt; the strong base includes sodium hydroxide and / or potassium hydroxide; the soluble calcium salt includes one or more combinations of calcium chloride, calcium nitrate, calcium acetate, calcium propionate, calcium lactate, and calcium gluconate.

3. The method for preparing pufferfish skin collagen peptides according to claim 2, characterized in that, The stirring speed is 30~80 r / min, 80~100 r / min or 100~150 r / min; The agitation time is 2~2.5 h, 2.5~3.5 h, or 3.5~4.0 h; The pH value of the pufferfish skin after alkali treatment is 6.5~7.

5.

4. The method for preparing pufferfish skin collagen peptides according to claim 1, characterized in that, In step B, The acidic extract is one or more combinations of citrate-sodium citrate buffer, acetate buffer, or hydrochloric acid solution; The acidic extract contains 0.04 to 0.06 mol / L sodium chloride, and the pH value of the acidic extract is 4.0 to 5.

0.

5. The method for preparing pufferfish skin collagen peptides according to claim 4, characterized in that, The mass-to-volume ratio of the alkali-treated pufferfish skin to the acidic extract is 1:(5~8), 1:(8~12), or 1:(12~15); The inert gas is one or more combinations of nitrogen, argon, and helium.

6. The method for preparing pufferfish skin collagen peptides according to claim 5, characterized in that, The temperature of the hydrothermal reaction is 70~75℃, 75~85℃ or 85~90℃; the time of the hydrothermal reaction is 4~5 h, 5~7 h or 7~8 h. Step B does not include enzymatic hydrolysis.

7. The method for preparing pufferfish skin collagen peptides according to claim 1, characterized in that, In step C The filter screen has a mesh size of 300-500. The dialysis cutoff is 3000-5000 Daltons, 5000-7000 Daltons, or 7000-10000 Daltons; The vacuum freeze-drying temperature is -60~-80℃, and the vacuum freeze-drying time is 24~48 h.

8. A cosmetic product, characterized in that, The cosmetic product includes pufferfish skin collagen peptides prepared by the method for preparing pufferfish skin collagen peptides according to any one of claims 1 to 7. The cosmetic is used for moisturizing or metabolic repair of connective tissue.

9. A medical dressing, characterized in that, The medical dressing comprises pufferfish skin collagen peptide prepared by the method of preparing pufferfish skin collagen peptide according to any one of claims 1 to 7; The medical dressing is used to absorb wound exudate or for wound repair.

10. A food product, characterized in that, The food product includes pufferfish skin collagen peptides prepared by the method for preparing pufferfish skin collagen peptides according to any one of claims 1 to 7. The food is intended to supplement calcium, promote intestinal absorption, or promote the metabolic repair of connective tissue.

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