Collagen peptide from swim bladder and preparation method and application thereof

Abstract

The application belongs to the technical field of proteins, and particularly relates to fish swim bladder collagen protein peptide and a preparation method and application thereof. The application provides a preparation method of fish swim bladder collagen protein peptide. The method adopts a composite natural eutectic solvent for collagen protein, elastin and chondroitin sulfate-proteoglycan complex structure, and is supplemented with ultrasonic treatment, so that synchronous and efficient extraction of collagen protein, elastin and chondroitin sulfate is realized. Further combined with step-by-step enzymatic hydrolysis of proteins, fish swim bladder collagen protein peptide rich in elastin peptide and chondroitin sulfate is prepared. The above method realizes simultaneous extraction of collagen protein peptide, elastin peptide and chondroitin sulfate from fish swim bladder, avoids resource waste caused by traditional single component development, has high value of the obtained product, and thus realizes high-value comprehensive utilization of fish swim bladder resources.

Description

Technical Field

[0001] This application belongs to the field of protein technology, specifically relating to fish swim bladder collagen peptides, their preparation methods, and applications. Background Technology

[0002] The swim bladder, also known as the fish bladder or fish stomach, is an organ of bony fish, accounting for about 5% of the fish's body volume. The swim bladder wall is composed of connective tissue and elastic fibers, rich in protein, various vitamins, and trace elements such as calcium, zinc, iron, and selenium. It is a high-protein, low-fat food and medicinal tonic. Current research shows that the protein content of the swim bladder can be as high as 79%, while the fat content is only 0.2%-0.4%.

[0003] Currently, research and development on fish swim bladders mainly focus on the extraction of collagen and collagen peptides contained within them. For example, traditional techniques have reported methods for extracting collagen from fish swim bladders using eutectic solvents. Chinese patent application CN118126159A discloses "a green and efficient preparation method for fish swim bladder collagen," which uses a single eutectic solvent composed of urea and organic acids (such as acetic acid, formic acid, or lactic acid) for extraction at a low temperature of 4°C for a long time (12-36 hours), followed by dialysis and freeze-drying to obtain type I collagen powder.

[0004] However, the aforementioned traditional technologies and most current research still have the following shortcomings:

[0005] The single-component nature of the product leads to resource waste: Current technologies mostly focus on extracting single components (mainly collagen) from fish swim bladders, while fish swim bladders are also a natural and high-quality source of elastin and chondroitin sulfate. This single-component extraction method fails to achieve the high-value comprehensive utilization of fish swim bladder resources, resulting in the waste of other active ingredients.

[0006] Extraction methods are selective, making it difficult to extract multiple components simultaneously: different eutectic solvents have varying affinities and selectivities for different target components (such as amino acids and polysaccharides). Existing technologies using a single eutectic solvent system primarily target collagen dissolution, but are inefficient at extracting elastin rich in cross-linked bonds and chondroitin sulfate in the form of proteoglycan complexes, making it difficult to achieve simultaneous and efficient extraction of all three.

[0007] Therefore, developing a method for simultaneously and efficiently extracting collagen, elastin, and chondroitin sulfate from fish swim bladders and converting them into small molecule peptides with specific bioactivity is of great significance for realizing the high-value comprehensive utilization of fish swim bladder resources and increasing the added value of products. Summary of the Invention

[0008] Based on this, one embodiment of this application provides fish swim bladder collagen peptides, their preparation method, and their applications.

[0009] This application provides a method for preparing fish swim bladder collagen peptides, comprising:

[0010] Provide a swim bladder and pre-treat the swim bladder;

[0011] Pretreated fish swim bladders were placed in a composite natural eutectic solvent and extracted under ultrasonic conditions to prepare collagen extract.

[0012] Elastase was added to the collagen extract to perform the first enzymatic hydrolysis, and the first enzymatic hydrolysate was prepared.

[0013] A complex protease is added to the first-step enzymatic hydrolysate for a second-step enzymatic hydrolysis to prepare the hydrolysate; and

[0014] The enzymatic hydrolysis product is subjected to enzyme inactivation treatment to prepare fish swim bladder collagen peptides;

[0015] The composite natural eutectic solvent includes a first natural eutectic solvent and a second natural eutectic solvent;

[0016] The first natural eutectic solvent includes betaine, lactic acid, and water;

[0017] The second natural eutectic solvent includes betaine, urea, and water.

[0018] In some embodiments, the molar ratio of betaine, lactic acid and water in the first natural eutectic solvent is 1:(2-4):(1-3).

[0019] The molar ratio of betaine, urea and water in the second natural eutectic solvent is 1:(2-5):(1-2).

[0020] In some embodiments, the volume ratio of the first natural eutectic solvent to the second natural eutectic solvent is (5-7):(3-5).

[0021] In some embodiments, the ratio of the fish bladder to the composite natural eutectic solvent is 1:(15-25)(g / mL).

[0022] In some embodiments, the ultrasound power is 500W-700W and the duration is 5-20 minutes.

[0023] This application also provides a method for preparing the fish swim bladder collagen peptides, wherein the complex protease includes two or more of alkaline protease, papain, bromelain, neutral protease, flavor protease, trypsin, and pepsin.

[0024] In some embodiments, the conditions for the first step of enzymatic hydrolysis include: the mass of the elastase is 0.5%-2.5% of the mass of the fish swim bladder, the pH is 8.5-9.5, the temperature is 50℃-65℃, and the time is 2h-4h.

[0025] In some embodiments, the conditions for the second enzymatic hydrolysis step include: the mass of the complex protease is 1%-4% of the mass of the fish swim bladder, the pH is 7.5-8.5, the temperature is 50℃-60℃, and the time is 3h-5h.

[0026] In some embodiments, the amount of elastase added is 0.6%-0.8% of the fish bladder mass.

[0027] In some embodiments, the amount of the complex protease added is 1.4%-1.6% of the fish swim bladder mass.

[0028] In some embodiments, the complex protease is an alkaline protease, a neutral protease, and a flavor protease.

[0029] In some embodiments, the mass ratio of the alkaline protease, the neutral protease, and the flavor protease is 1:(0.5-1):(0.2-0.5).

[0030] In some embodiments, the enzyme inactivation treatment is followed by separation and purification treatment and drying treatment.

[0031] In some embodiments, the separation and purification process includes a first filtration using an ultrafiltration membrane with a molecular weight cutoff of 8 kDa-12 kDa, followed by a second filtration using a nanofiltration membrane with a molecular weight cutoff of 180 Da-220 Da.

[0032] In some of these embodiments, the drying process includes spray drying.

[0033] In some embodiments, the parameters for spray drying include an outlet air temperature of 85°C-90°C and an inlet air temperature of 170°C-190°C.

[0034] This application also provides a fish swim bladder collagen peptide, which is prepared using the aforementioned method for preparing fish swim bladder collagen peptides.

[0035] This application also provides the use of the aforementioned fish maw collagen peptides in the preparation of products that promote bone health and resist photodamage to the skin.

[0036] This application provides a method for preparing fish swim bladder collagen peptides. The method employs a composite natural eutectic solvent targeting the structures of collagen, elastin, and chondroitin sulfate-proteoglycan complexes, supplemented by ultrasonic treatment, to achieve simultaneous and efficient extraction of collagen, elastin, and chondroitin sulfate. Further, stepwise enzymatic hydrolysis of proteins is used to obtain fish swim bladder collagen peptides rich in elastin peptides and chondroitin sulfate. This method achieves the simultaneous extraction of collagen peptides, elastin peptides, and chondroitin sulfate from fish swim bladders, avoiding the resource waste caused by traditional single-component development. The resulting product has high value, thus realizing the high-value comprehensive utilization of fish swim bladder resources. Attached Figure Description

[0037] To more clearly illustrate the technical solutions in the embodiments of this application and to more completely understand this application and its beneficial effects, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0038] Figure 1 The CCK-8 experimental results show the effect of the fish swim bladder collagen peptide prepared in Example 1 of this application on the proliferation of different cells (osteoblasts and fibroblasts).

[0039] Figure 2 Alizarin Red staining results for the effect of fish swim bladder collagen peptides prepared in Example 1 of this application on the formation of mineralized nodules in osteoblasts;

[0040] Figure 3 The image shows the alkaline phosphatase (ALP) staining results of the effect of fish swim bladder collagen peptides prepared in Example 1 of this application on early osteoblast differentiation.

[0041] Figure 4 The figure shows the Western Blot results of the effect of the fish swim bladder collagen peptide prepared in Example 1 of this application on the expression levels of type I collagen (Collagen I) and matrix metalloproteinase 1 (MMP-1) in fibroblasts. Detailed Implementation

[0042] The present application will be further described in detail below with reference to the embodiments and examples. It should be understood that these embodiments and examples are for illustrative purposes only and are not intended to limit the scope of the present application. The purpose of providing these embodiments and examples is to enable a more thorough and comprehensive understanding of the disclosure of the present application. It should also be understood that the present application can be implemented in many different forms and is not limited to the embodiments and examples described herein. Those skilled in the art can make various modifications or alterations without departing from the spirit of the present application, and the equivalent forms obtained also fall within the protection scope of the present application. Furthermore, numerous specific details are set forth in the following description to provide a fuller understanding of the present application. It should be understood that the present application can be implemented without one or more of these details.

[0043] Unless otherwise defined, all technical and scientific terms used in this application have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0044] Unless otherwise stated or in case of contradiction, the terms or phrases used herein shall have the following meanings:

[0045] The terms "and / or," "or / and," and "and / or" as used herein include any one of two or more of the related listed items, as well as any and all combinations of the related listed items. These arbitrary and all combinations include any two related listed items, any more related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected by at least two conjunctions selected from "and / or," "or / and," and "and / or," it should be understood that in this application, the technical solution undoubtedly includes technical solutions connected by "logical AND," and also undoubtedly includes technical solutions connected by "logical OR." For example, "A and / or B" includes three parallel solutions: A, B, and A+B. For example, the technical solution of "A, and / or, B, and / or, C, and / or, D" includes any one of A, B, C, and D (that is, a technical solution that is connected by "logical OR"), as well as any and all combinations of A, B, C, and D, that is, combinations of any two or three of A, B, C, and D, and also combinations of all four of A, B, C, and D (that is, a technical solution that is connected by "logical AND").

[0046] In this application, the terms "multiple", "various", "multiple times", "multi-dimensional", etc., unless otherwise specified, refer to a quantity greater than or equal to 2. For example, "one or more" means one or more than or equal to two.

[0047] The terms “combinations of,” “any combination of,” and “any combination of” used in this article include all suitable combinations of any two or more of the listed items.

[0048] In this document, the term "suitable" as used in phrases such as "suitable combination," "suitable method," and "any suitable method" refers to the ability to implement the technical solution of this application, solve the technical problem of this application, and achieve the expected technical effect of this application.

[0049] In this application, terms such as "further," "even more," and "particularly" are used for descriptive purposes and to indicate differences in content, but should not be construed as limiting the scope of protection of this application.

[0050] In this application, "optionally," "optionally," and "optional" mean that something is optional, that is, it means that it is selected from either "with" or "without." If there are multiple "optional" entries in a technical solution, unless otherwise specified, and there are no contradictions or mutual constraints, each "optional" entry shall be independent.

[0051] In this application, the technical features described in an open-ended manner include both closed technical solutions composed of the listed features and open technical solutions composed of the listed features.

[0052] In this application, numerical intervals (i.e., numerical ranges) are involved. Unless otherwise specified, the selected numerical distributions within the aforementioned numerical intervals are considered continuous and include the two endpoints (i.e., the minimum and maximum values) of the numerical range, as well as every value between these two endpoints. Unless otherwise specified, when a numerical interval refers only to integers within that interval, it includes the two endpoint integers of the numerical range, as well as every integer between the two endpoints. In this document, this is equivalent to directly listing every integer. For example, if t is an integer selected from 1 to 10, it means that t is any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Furthermore, when multiple ranges are provided to describe features or characteristics, these ranges can be merged. In other words, unless otherwise specified, the ranges disclosed herein should be understood to include any and all subranges to which they are included.

[0053] Unless otherwise specified, the temperature parameters in this application are permitted to be either constant-temperature treatment or variations within a certain temperature range. It should be understood that the constant-temperature treatment allows temperature fluctuations within the precision range of the instrument control, such as ±5℃, ±4℃, ±3℃, ±2℃, or ±1℃.

[0054] In this application, % (w / w) and wt% both represent weight percentage, % (v / v) refers to volume percentage, and % (w / v) refers to mass-volume percentage.

[0055] All references to documents mentioned in this application are incorporated herein by reference as if each document were individually incorporated herein by reference. Unless they conflict with the inventive purpose and / or technical solution of this application, all cited documents are incorporated herein by reference in their entirety and for all purposes. When citing documents in this application, the definitions of relevant technical features, terms, nouns, phrases, etc., are also incorporated herein by reference. When citing documents in this application, examples and preferred embodiments of the cited technical features may also be incorporated herein by reference, but only to the extent that they enable the implementation of this application. It should be understood that when the cited content conflicts with the description in this application, this application shall prevail or modifications shall be made adaptably to the description in this application.

[0056] This application provides a method for preparing fish swim bladder collagen peptides, comprising:

[0057] Provide a swim bladder and pre-treat the swim bladder;

[0058] Pretreated fish swim bladders were placed in a composite natural eutectic solvent and extracted under ultrasonic conditions to prepare collagen extract.

[0059] Elastase was added to the collagen extract to perform the first enzymatic hydrolysis, and the first enzymatic hydrolysate was prepared.

[0060] A complex protease is added to the first-step enzymatic hydrolysate for a second-step enzymatic hydrolysis to prepare the hydrolysate; and

[0061] The enzymatic hydrolysis product is subjected to enzyme inactivation treatment to prepare fish swim bladder collagen peptides;

[0062] The composite natural eutectic solvent includes a first natural eutectic solvent and a second natural eutectic solvent;

[0063] The first natural eutectic solvent includes betaine, lactic acid, and water;

[0064] The second natural eutectic solvent includes betaine, urea, and water.

[0065] This application uses a complex natural eutectic solvent for extraction, with different components targeting the unique structures of collagen (easily hydrolyzed by acid / enzyme), elastin (rich in cross-links and requiring selective cleavage), and chondroitin sulfate-proteoglycan complex (requiring disruption of ionic and hydrogen bonds). Ultrasonic assistance is employed to achieve simultaneous and efficient extraction.

[0066] In some embodiments, the molar ratio of betaine, lactic acid, and water in the first natural eutectic solvent is 1:(2-4):(1-3). For example, the molar ratio of betaine, lactic acid, and water in the first natural eutectic solvent is 1:(2, 3, 4):(1, 2, 3).

[0067] In some embodiments, the molar ratio of betaine, urea, and water in the second natural eutectic solvent is 1:(2-5):(1-2). For example, the molar ratio of betaine, urea, and water in the second natural eutectic solvent is 1:(2, 3, 4, 5):(1, 2, 3).

[0068] The above ratio gives the solvent an appropriate hydrogen bond network structure and viscosity, which can effectively disrupt the triple helix structure of collagen, the cross-linking bonds of elastin, and the ionic bonds of the chondroitin sulfate-proteoglycan complex without excessively damaging the active structure of the target product, thereby achieving simultaneous and efficient extraction of collagen, elastin, and chondroitin sulfate from fish swim bladders.

[0069] In some embodiments, the volume ratio of the first natural eutectic solvent to the second natural eutectic solvent is (5-7):(3-5) or any value in between. For example, the volume ratio of the first natural eutectic solvent to the second natural eutectic solvent is (5, 6, 7):(3, 4, 5) or any value in between. Mixing the first and second natural eutectic solvents within this volume ratio range ensures efficient extraction of collagen while also facilitating the dissolution of elastin and chondroitin sulfate.

[0070] In some embodiments, the ratio of the swim bladder to the composite natural eutectic solvent is 1:(15-25) g / mL. The ratio of the swim bladder to the composite natural eutectic solvent is 1:15, 1:16, 1:17, 1:18, 1:19, 1:20, 1:21, 1:22, 1:23, 1:24, or 1:25, or any value in between.

[0071] In some embodiments, the ultrasonic power is 500W-700W, and the duration is 5min-20min. For example, the ultrasonic power can be 500W, 520W, 540W, 560W, 580W, 600W, 620W, 640W, 660W, 680W, or 700W, or any value in between. The duration can be 5min, 6min, 7min, 8min, 9min, 10min, 11min, 12min, 13min, 14min, 15min, 16min, 17min, 18min, 19min, or 20min. Utilizing the cavitation and mechanical effects of ultrasound, fish swim bladder tissue can be effectively broken down, the complex natural eutectic solvent can be penetrated into the tissue, and the dissolution of collagen, elastin, and chondroitin sulfate can be accelerated.

[0072] In some embodiments, the stepwise enzymatic hydrolysis process includes: adding 0.5%-2.5% elastase by weight of fish swim bladder to the collagen extract, adjusting the pH to 8.5-9.5, and hydrolyzing at 50-65°C for 2-4 hours to prepare the first-step hydrolysate; for example, the amount of elastase (by weight of fish swim bladder) is 0.5%, 1.0%, 1.5%, 2.0%, or 2.5%, or any value in between. For example, the pH of the first-step hydrolysis is 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, or 9.5, or any value in between. For example, the temperature of the first-step hydrolysis is 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C, 60°C, 61°C, 62°C, 63°C, 64°C, or 65°C, or any value in between. For example, the first step of enzymatic hydrolysis time can be 2h, 3h, or 4h, or any value in between.

[0073] Add 1%-4% of the fish swim bladder mass of the first step enzymatic hydrolysate to the hydrolysate, adjust the pH to 7.5-8.5, and enzymatically hydrolyze at 50-60℃ for 3-5 hours to prepare the fish swim bladder extract hydrolysate;

[0074] For example, the dosage of the complex protease (as a percentage of fish swim bladder mass): 1%, 2%, 3%, or 4%, or any value in between. For example, the pH for the second step of enzymatic hydrolysis: 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or 8.5, or any value in between. For example, the temperature for the second step of enzymatic hydrolysis: 50℃, 51℃, 52℃, 53℃, 54℃, 55℃, 56℃, 57℃, 58℃, 59℃, or 60℃, or any value in between. For example, the time for the second step of enzymatic hydrolysis: 3h, 4h, or 5h, or any value in between.

[0075] The complex protease includes two or more of the following: alkaline protease, papain, bromelain, neutral protease, flavor protease, trypsin, and pepsin.

[0076] In some embodiments, the amount of elastase added is 0.6%-0.8% of the swim bladder mass; for example, the amount of elastase added is 0.6%, 0.7% or 0.8% of the swim bladder mass, or any value in between.

[0077] In some embodiments, the amount of complex protease added is 1.4%-1.6% of the swim bladder mass; for example, the amount of complex protease added is 1.4%, 1.5% or 1.6% of the swim bladder mass and any value in between.

[0078] In some embodiments, the complex protease is an alkaline protease, a neutral protease, and a flavor protease;

[0079] In some embodiments, the mass ratio of the alkaline protease, the neutral protease, and the flavor protease is 1:(0.5-1):(0.2-0.5). The mass ratio of the alkaline protease, the neutral protease, and the flavor protease is 1:(0.5, 0.6, 0.7, 0.8, 0.9, 1):(0.2, 0.3, 0.4, 0.5) and any values ​​in between.

[0080] In some embodiments, the enzyme inactivation treatment is followed by separation and purification treatment and drying treatment.

[0081] In some embodiments, the separation and purification process includes a first filtration using an ultrafiltration membrane of 8 kDa-12 kDa, followed by a second filtration using a nanofiltration membrane of 180 Da-220 Da; for example, the ultrafiltration membrane has a molecular weight cutoff of 8 kDa, 9 kDa, 10 kDa, 11 kDa, or 12 kDa, or any value in between; for example, the nanofiltration membrane has a molecular weight cutoff of 180 Da, 190 Da, 200 Da, 210 Da, or 220 Da, or any value in between.

[0082] In some embodiments, the drying process includes spray drying, wherein the parameters of the spray drying include an outlet air temperature of 85-90°C and an inlet air temperature of 170-190°C. For example, the spray drying outlet air temperature can be 85°C, 86°C, 87°C, 88°C, 89°C, or 90°C, or any value in between. For example, the spray drying inlet air temperature can be 170°C, 175°C, 180°C, 185°C, or 190°C, or any value in between.

[0083] This application also provides a fish swim bladder collagen peptide, which is prepared using the aforementioned method for preparing fish swim bladder collagen peptides.

[0084] This application also provides the use of the aforementioned fish maw collagen peptides in the preparation of products that promote bone health and resist photodamage to the skin.

[0085] The fish swim bladder collagen peptides prepared in this application can improve bone health by promoting osteoblast proliferation and differentiation. In addition, they also have the bioactivity of promoting type I collagen expression and inhibiting MMP-1 overexpression, exerting a potential anti-photodamage effect, and can effectively promote bone health and resist skin photodamage.

[0086] The embodiments of this application will be described in detail below with reference to examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of this application. For experimental methods in the following embodiments where specific conditions are not specified, please refer to the guidelines given in this application, or follow experimental manuals or conventional conditions in the art, or follow the conditions recommended by the manufacturer, or refer to experimental methods known in the art.

[0087] In the specific embodiments described below, the measurement parameters involving raw material components may have slight deviations within the weighing accuracy range unless otherwise specified. Temperature and time parameters are subject to acceptable deviations due to instrument testing accuracy or operational precision.

[0088] It should be understood that in the various embodiments of this application, the order of the above-mentioned processes does not imply the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0089] Example 1

[0090] This embodiment provides a method for preparing fish swim bladder collagen peptides, comprising the following steps:

[0091] 1. Raw material pretreatment: Thaw the swim bladder of silver carp, clean it and cut it into pieces.

[0092] 2. Extraction with composite natural eutectic solvent (NADES): The pretreated fish bladder was mixed with the composite natural eutectic solvent and reacted at 55℃ (constant temperature water bath) for 45 min. The ratio of fish bladder to composite natural eutectic solvent was 1:20 (g / mL).

[0093] Preparation method of composite natural eutectic solvent: First natural eutectic solvent: Betaine, lactic acid, and water are mixed in a molar ratio of 1:2:1 and stirred at 60°C until the solution is clear and transparent; Second natural eutectic solvent: Betaine, urea, and water are mixed in a molar ratio of 1:2:1.5 and stirred in a water bath at 55°C until clear. The first and second natural eutectic solvents are mixed at a volume ratio of 7:3 to obtain the composite natural eutectic solvent.

[0094] 3. Ultrasonic-assisted extraction: The extraction container is subjected to ultrasonic treatment at a power of 600W for 10 minutes.

[0095] 4. Enzymatic hydrolysis: Step 1: Add 0.8% elastase (by weight of fish bladder) to the swim bladder, adjust the pH to 8.5, and hydrolyze at 55℃ for 2 hours to obtain the first step swim bladder extract hydrolysate. Step 2: Add 1.5% complex protease (by weight of fish bladder) to the first step swim bladder extract hydrolysate, adjust the pH to 8.0, and hydrolyze at 60℃ for 3.5 hours to obtain the swim bladder extract hydrolysate. The complex protease consists of alkaline protease, neutral enzyme, and flavor enzyme in a mass ratio of 1:0.5:0.2.

[0096] 5. Inactivate enzymes: Heat the enzyme hydrolysate at 95°C for 15 minutes, then cool to room temperature.

[0097] 6. Centrifugation: Centrifuge at 6000 r / min for 15 min, discard the precipitate, and obtain the supernatant.

[0098] 7. Separation and purification: The supernatant is separated using an ultrafiltration membrane (10 kDa) to remove large molecular weight peptides and impurities, resulting in a small molecular weight peptide solution. This solution is then filtered through a nanofiltration membrane with a molecular weight cutoff of 200 Da to remove water, monovalent salts, and free amino acids.

[0099] 8. Spray drying: The outlet air temperature is 80-90℃, and the inlet air temperature is 180℃.

[0100] Example 2

[0101] This embodiment provides a method for preparing fish swim bladder collagen peptides, comprising the following steps:

[0102] 1. Raw material pretreatment: Thaw the fish maw, clean it and cut it into pieces.

[0103] 2. Extraction with a compound natural eutectic solvent: The pretreated fish bladder was mixed with a compound natural eutectic solvent and reacted at 55℃ (constant temperature water bath) for 45 min. The ratio of fish bladder to compound natural eutectic solvent was 1:20 (g / mL).

[0104] Preparation method of composite natural eutectic solvent: First natural eutectic solvent: Betaine, lactic acid, and water are mixed in a molar ratio of 1:3:1 and stirred at 60°C until the solution is clear and transparent; Second natural eutectic solvent: Betaine, urea, and water are mixed in a molar ratio of 1:2:1.5 and stirred in a water bath at 55°C until clear. The first and second natural eutectic solvents are mixed at a volume ratio of 7:3 to obtain the composite natural eutectic solvent.

[0105] 3. Ultrasonic-assisted extraction: The extraction container is subjected to ultrasonic treatment at a power of 600W for 15 minutes.

[0106] 4. Enzymatic hydrolysis: Step 1: Add 0.8% elastase (by weight of fish bladder) to the swim bladder, adjust the pH to 8.5, and hydrolyze at 55℃ for 2 hours to obtain the first step swim bladder extract hydrolysate. Step 2: Add 1.5% complex protease (by weight of fish bladder) to the first step swim bladder extract hydrolysate, adjust the pH to 8.0, and hydrolyze at 60℃ for 3.5 hours to obtain the swim bladder extract hydrolysate. The complex protease consists of alkaline protease, neutral enzyme, and flavor enzyme in a mass ratio of 1:0.5:0.2.

[0107] 5. Inactivate enzymes: Heat the enzyme hydrolysate at 95°C for 15 minutes, then cool to room temperature.

[0108] 6. Centrifugation: Centrifuge at 6000 r / min for 15 min, discard the precipitate, and obtain the supernatant.

[0109] 7. Separation and purification: The supernatant is separated using an ultrafiltration membrane (10 kDa) to remove large molecular weight peptides and impurities, resulting in a small molecular weight peptide solution. This solution is then filtered through a nanofiltration membrane with a molecular weight cutoff of 200 Da to remove water, monovalent salts, and free amino acids.

[0110] 8. Spray drying: outlet air temperature is 90℃, inlet air temperature is 180℃.

[0111] Example 3

[0112] This embodiment provides a method for preparing fish swim bladder collagen peptides, comprising the following steps:

[0113] 1. Raw material pretreatment: Thaw the fish maw, clean it and cut it into pieces.

[0114] 2. Extraction with a compound natural eutectic solvent: The pretreated fish bladder was mixed with a compound natural eutectic solvent and reacted at 55℃ (constant temperature water bath) for 45 min. The ratio of fish bladder to compound natural eutectic solvent was 1:20 (g / mL).

[0115] Preparation method of composite natural eutectic solvent: First natural eutectic solvent: Betaine, lactic acid, and water are mixed in a molar ratio of 1:2:1 and stirred at 60°C until the solution is clear and transparent; Second natural eutectic solvent: Betaine, urea, and water are mixed in a molar ratio of 1:2:1.5 and stirred in a water bath at 55°C until clear. The first and second natural eutectic solvents are mixed at a volume ratio of 6:4 to obtain the composite natural eutectic solvent.

[0116] 3. Ultrasonic-assisted extraction: The extraction container is subjected to ultrasonic treatment at a power of 600W for 20 minutes.

[0117] 4. Enzymatic hydrolysis: Step 1: Add 0.8% elastase (by weight of fish bladder) to the swim bladder, adjust the pH to 8.5, and hydrolyze at 55℃ for 2 hours to obtain the first step swim bladder extract hydrolysate. Step 2: Add 1.5% complex protease (by weight of fish bladder) to the first step swim bladder extract hydrolysate, adjust the pH to 8.0, and hydrolyze at 60℃ for 3.5 hours to obtain the swim bladder extract hydrolysate. The complex protease consists of alkaline protease, neutral enzyme, and flavor enzyme in a mass ratio of 1:0.5:0.2.

[0118] 5. Inactivate enzymes: Heat the enzyme hydrolysate at 95°C for 15 minutes, then cool to room temperature.

[0119] 6. Centrifugation: Centrifuge at 6000 r / min for 15 min, discard the precipitate, and obtain the supernatant.

[0120] 7. Separation and purification: The supernatant is separated using an ultrafiltration membrane (10 kDa) to remove large molecular weight peptides and impurities, resulting in a small molecular weight peptide solution. This solution is then filtered through a nanofiltration membrane with a molecular weight cutoff of 200 Da to remove water, monovalent salts, and free amino acids.

[0121] 8. Spray drying: outlet air temperature is 85℃, inlet air temperature is 180℃.

[0122] Example 4

[0123] This embodiment provides a method for preparing fish swim bladder collagen peptides, comprising the following steps:

[0124] 1. Raw material pretreatment: Thaw the fish maw, clean it and cut it into pieces.

[0125] 2. Extraction with a compound natural eutectic solvent: The pretreated fish bladder was mixed with the compound natural eutectic solvent and reacted at 55℃ (constant temperature water bath) for 45 min. The ratio of fish bladder to compound natural eutectic solvent was 1:20 (g / mL).

[0126] Preparation method of composite natural eutectic solvent: First natural eutectic solvent: Betaine, lactic acid, and water are mixed in a molar ratio of 1:2:1 and stirred at 60°C until the solution is clear and transparent; Second natural eutectic solvent: Betaine, urea, and water are mixed in a molar ratio of 1:2:1.5 and stirred in a water bath at 55°C until clear. The first and second natural eutectic solvents are mixed at a volume ratio of 7:3 to obtain the composite natural eutectic solvent.

[0127] 3. Ultrasonic-assisted extraction: The extraction container is subjected to ultrasonic treatment at a power of 600W for 5 minutes.

[0128] 4. Enzymatic hydrolysis: Step 1: Add 0.6% elastase (by weight of fish bladder) to the swim bladder, adjust the pH to 8.5, and hydrolyze at 55℃ for 2 hours to obtain the first step swim bladder extract hydrolysate. Step 2: Add 1.5% complex protease (by weight of fish bladder) to the first step swim bladder extract hydrolysate, adjust the pH to 8.0, and hydrolyze at 60℃ for 3.5 hours to obtain the swim bladder extract hydrolysate. The complex protease consists of alkaline protease, neutral enzyme, and flavor enzyme in a mass ratio of 1:0.5:0.2.

[0129] 5. Inactivate enzymes: Heat the enzyme hydrolysate at 95°C for 15 minutes, then cool to room temperature.

[0130] 6. Centrifugation: Centrifuge at 6000 r / min for 15 min, discard the precipitate, and obtain the supernatant.

[0131] 7. Separation and purification: The supernatant is separated using an ultrafiltration membrane (10 kDa) to remove large molecular weight peptides and impurities, resulting in a small molecular weight peptide solution. This solution is then filtered through a nanofiltration membrane with a molecular weight cutoff of 200 Da to remove water, monovalent salts, and free amino acids.

[0132] 8. Spray drying: outlet air temperature is 90℃, inlet air temperature is 180℃.

[0133] Comparative Example 1

[0134] Step 3 of Example 1 is deleted, and ultrasound-assisted extraction is not performed. Other conditions are the same as in Example 1.

[0135] Comparative Example 2

[0136] In step 2, the composite natural eutectic solvent was replaced with only the second natural eutectic solvent, while other conditions remained the same as in Example 1.

[0137] Comparative Example 3

[0138] In step 2, the composite natural eutectic solvent was replaced with the first natural eutectic solvent, while other conditions remained the same as in Example 1.

[0139] Comparative Example 4

[0140] In step 2, both the first and second natural eutectic solvents in the composite natural eutectic solvent are betaine, lactic acid, and water.

[0141] Comparative Example 5

[0142] In step 2, both the first and second natural eutectic solvents in the composite natural eutectic solvent are betaine, urea, and water.

[0143] Comparative Example 6

[0144] The stepwise enzymatic hydrolysis in step 4 was changed to a one-step enzymatic hydrolysis. Simultaneously, 0.6% elastase and 1.5% complex protease (by weight of fish swim bladder) were added, the pH was adjusted to 8.5, and enzymatic hydrolysis was carried out at 60℃ for 5.5 h to obtain the fish swim bladder extract hydrolysate. The complex protease consisted of alkaline protease, neutral enzyme, and flavor enzyme in a mass ratio of 1:0.5:0.2. Other conditions were the same as in Example 1.

[0145] Result verification:

[0146] I. The yield, hydroxyproline content, desmodium + isodesmodium content and chondroitin sulfate content of each embodiment and comparative example are shown in Table 1.

[0147] The enzymes involved in this application are all commercially available proteases. The alkaline protease (enzyme activity: 200,000 U / g), trypsin (enzyme activity: 200,000 U / g), and bromelain (enzyme activity: 100,000 U / g) are from Cangzhou Xiasheng Enzyme Biotechnology Co., Ltd.; papain (enzyme activity: 400,000) is from Nanning Pangbo Bioengineering Co., Ltd.; neutral protease (enzyme activity: 1600 AU / g) is from Genentech Co., Ltd.; pepsin (product number: P8160, enzyme activity: ≥250 u / mg) is from Beijing Solarbio Technology Co., Ltd.; and flavor protease (enzyme activity unit: 200,000 U / g) is from Novozymes (China) Biotechnology Co., Ltd.

[0148] This application utilizes an acid hydrolysis method and a hydroxyproline kit to determine the hydroxyproline content in fish swim bladder peptides.

[0149] The determination method for the content of desmokine and isodesmokine in this application refers to T / CHC1010-2023 "Elastin Peptides", Appendix A.

[0150] The method for determining chondroitin sulfate in this application refers to GB / T 20365-2006 "Determination of chondroitin sulfate and glucosamine hydrochloride content by liquid chromatography".

[0151] Table 1: Yield, hydroxyproline content, and chondroitin sulfate content of each example

[0152]

[0153] As shown in Table 1, the yields of Examples 1-4 were all higher than 15%, the hydroxyproline content was all greater than 9%, the sum of desmodium and isodesmodium content was all higher than 0.05%, and the chondroitin sulfate content was all higher than 1.3%. The yield and content of each component of Comparative Example 2 were similar to those of Comparative Example 5, but the hydroxyproline content was significantly lower than that of Example 1, indicating that the extraction effect of collagen by using the second natural eutectic solvent alone was not good. The yield and content of each component of Comparative Example 3 were not significantly different from those of Comparative Example 4, but the desmodium and isodesmodium content was significantly lower than that of Example 1, indicating that the extraction effect of elastin by using the first natural eutectic solvent alone was not good. The yield, hydroxyproline, desmodium and isodesmodium content of Comparative Example 6 were all significantly lower than those of Example 1, indicating that the enzymatic hydrolysis effect of stepwise enzymatic hydrolysis was significantly better than that of one-step enzymatic hydrolysis.

[0154] II. Experiments were conducted using fish swim bladder collagen peptide samples rich in elastin peptides and chondroitin sulfate from Example 1.

[0155] 1. Effects of fish swim bladder peptides on osteoblast proliferation

[0156] Methods: Osteoblasts and fibroblasts in logarithmic growth phase were seeded into 96-well plates, with a certain amount of cell suspension in each well. The plates were incubated at 37°C with 5% CO2 for 24 h to allow cell adhesion. After cell adhesion, different concentrations of fish swim bladder peptide solution (1 mg / mL, 2 mg / mL, 3 mg / mL, and 4 mg / mL) were added, and a blank control group was also included. After 12 h of further incubation, CCK-8 reagent was added to each well. Following incubation, the absorbance of each well was measured at 450 nm using a microplate reader.

[0157] result: Figure 1 The results showed that in osteoblasts, the proliferation rates of all treatment groups were slightly higher than those of the control group, but the overall difference was small. With increasing treatment concentration, the proliferation rate showed a slight upward trend, reaching a relative peak at 3 mg / mL, and then slightly decreased at 4 mg / mL, but remained higher than the control level. Similarly, in fibroblasts, the proliferation rates of cells treated with different concentrations of fish maw peptides were slightly higher than those of the control group. The proliferation rate of the 3 mg / mL group was significantly higher than that of the other concentration groups, while the proliferation rates of the 1 mg / mL and 2 mg / mL groups were relatively similar and slightly lower than those of the 3 mg / mL group. In summary, fish maw peptides showed a certain promoting effect on the proliferation of both osteoblasts and fibroblasts.

[0158] 2. Effects of fish swim bladder peptides on osteoblast mineralization

[0159] Methods: Osteoblasts in good growth condition were seeded into 24-well plates and cultured at 37℃ in a 5% CO2 incubator for 24 h to allow cell adhesion. Subsequently, experimental groups with different concentrations of fish swim bladder peptide (1 mg / mL, 2 mg / mL, 3 mg / mL, and 4 mg / mL) and a blank control group (containing only cell culture medium) were established. After replacing the culture medium with the corresponding concentration of fish swim bladder peptide, the cells were cultured for another 12 h. After culture, osteoblast mineralization nodules were detected using an osteoblast mineralization nodule staining kit, and the formation of mineralization nodules was observed using Alizarin Red staining.

[0160] Results: Alizarin Red staining results are as follows Figure 2 As shown, different concentrations of fish bladder peptides significantly affected the formation of osteoblast mineralized nodules. Compared with the control group, all experimental groups showed varying degrees of enhanced red staining, indicating that fish bladder peptides can promote osteoblast mineralization. As the concentration of fish bladder peptides increased from 1 mg / mL to 3 mg / mL, the number of mineralized nodules and the staining intensity gradually increased, showing a certain concentration dependence. The 3 mg / mL group showed the most obvious staining, with a large number and dense distribution of mineralized nodules, indicating its strongest effect in promoting osteoblast mineralization. When the concentration increased to 4 mg / mL, although the mineralization level was still higher than the control group, it did not show further enhancement compared to the 3 mg / mL group, indicating that the mineralization-promoting effect of high-concentration fish bladder peptides tended to plateau. These results indicate that fish bladder peptides can effectively promote the formation of osteoblast mineralized nodules, and its optimal concentration may be 3 mg / mL.

[0161] 3. Effects of fish swim bladder peptides on osteoblast differentiation

[0162] Methods: Cells were seeded in 24-well plates. When the cells reached 80% confluence, the medium was replaced with different concentrations of fish swim bladder peptide (0 mg / mL, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL) to induce differentiation. The cells were then incubated at 37°C in a 5% CO2 incubator for 12 h. Alkaline phosphatase (ALP) activity was detected using an alkaline phosphatase (ALP) kit to reflect the early differentiation of osteoblasts.

[0163] result: Figure 3ALP staining results showed that different concentrations of fish bladder peptides significantly affected the early differentiation of osteoblasts. Compared with the control group, all experimental groups showed varying degrees of enhanced blue-purple positive staining, indicating that fish bladder peptides could increase ALP activity in osteoblasts. As the concentration of fish bladder peptides increased from 1 mg / mL to 3 mg / mL, the number of ALP-positive cells and the staining intensity gradually increased, showing a clear concentration dependence. The staining was most significant in the 3 mg / mL group, indicating that this concentration had the strongest promoting effect on early osteoblast differentiation. When the concentration was further increased to 4 mg / mL, although the ALP activity was still higher than that in the control group, it was not significantly enhanced compared to the 3 mg / mL group, indicating that its differentiation-promoting effect tended to plateau. In conclusion, fish bladder peptides can effectively promote early osteoblast differentiation and exhibit the best promoting effect within a certain concentration range.

[0164] 4. Effects of fish swim bladder peptides on the expression levels of type I collagen and MMP1 in fibroblasts

[0165] Methods: Western blotting was used to detect the expression levels of type I collagen (Collagen I) and MMP1 protein in fibroblasts treated with different concentrations of fish swim bladder peptide. Fibroblasts in the logarithmic growth phase were seeded into culture plates. When the cell confluence reached approximately 80%, fish swim bladder peptide solutions with final concentrations of 1 mg / mL, 2 mg / mL, 3 mg / mL, and 4 mg / mL were added, respectively. A blank control group was also included. After 12 h of culture, cells were collected, total protein was extracted, and protein concentration was determined using the BCA method to ensure consistent loading amounts. SDS-PAGE electrophoresis was then performed, and the separated proteins were transferred to PVDF membranes. The membranes were blocked with 5% bovine serum albumin solution at room temperature for 2 h. Then, type I collagen primary antibody (catalog number 72026S, purchased from Cell Signaling Technology) and MMP1 primary antibody (catalog number 26585-1-AP, purchased from Wuhan Sanying Biotechnology Co., Ltd.) were added at a dilution of 1:1000, and the membranes were incubated overnight at 4°C. The next day, the membranes were washed three times with TBST for 10 min each time, and then a secondary antibody (diluted 1:100000) was added and incubated at room temperature for 2 h. After washing, chemiluminescence was used for color development, and images were acquired using a gel imaging system and grayscale analysis was performed. GAPDH was used as an internal control to calculate the relative expression levels of type I collagen and MMP1 proteins in each group.

[0166] Result: As Figure 4As shown in the figure, compared with the blank control group, UV treatment significantly decreased Collagen I expression in fibroblasts, while MMP-1 protein expression significantly increased, indicating that UV treatment induced changes related to extracellular matrix degradation. After intervention with different concentrations of fish bladder peptides, the expression level of type I collagen generally showed a recovery trend, with all treatment groups showing an increase compared to the UV group. The 1 mg / mL and 4 mg / mL groups showed the most significant increases, but overall, the levels remained lower than the blank control, indicating that fish bladder peptides can alleviate the inhibitory effect of UV on collagen synthesis to some extent. Meanwhile, MMP-1 protein expression was significantly upregulated in the UV group, while fish bladder peptide treatment showed varying degrees of decrease, with the 2 mg / mL group showing the most significant inhibitory effect. Subsequently, MMP-1 expression slightly recovered with increasing concentration, but remained lower than the UV group level.

[0167] In conclusion, fish swim bladder peptide intervention can, to some extent, reverse the UV-induced metabolic imbalance of fibroblast matrix and exert a potential anti-photodamage effect by promoting type I collagen expression and inhibiting MMP-1 overexpression.

[0168] The embodiments described above are merely illustrative of several implementation methods of this application, intended to facilitate a detailed understanding of the technical solutions of this application, but should not be construed as limiting the scope of protection of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Furthermore, it should be understood that after reading the above teachings of this application, those skilled in the art can make various alterations or modifications to this application, and the equivalent forms obtained also fall within the scope of protection of this application. It should also be understood that technical solutions obtained by those skilled in the art based on the technical solutions provided in this application through logical analysis, reasoning, or limited experimentation are all within the scope of protection of the appended claims. Therefore, the scope of protection of this patent application should be determined by the content of the appended claims, and the specification can be used to interpret the content of the claims.

Claims

1. A method for preparing fish swim bladder collagen peptides, characterized in that, include: Provide a swim bladder and pre-treat the swim bladder; Pretreated fish swim bladders were placed in a composite natural eutectic solvent and extracted under ultrasonic conditions to prepare collagen extract. Elastase was added to the collagen extract to perform the first enzymatic hydrolysis, and the first enzymatic hydrolysate was prepared. A complex protease is added to the first-step enzymatic hydrolysate to carry out the second-step enzymatic hydrolysis, thereby preparing the enzymatic hydrolysate; as well as The enzymatic hydrolysis product is subjected to enzyme inactivation treatment to prepare fish swim bladder collagen peptides; The composite natural eutectic solvent includes a first natural eutectic solvent and a second natural eutectic solvent; The first natural eutectic solvent includes betaine, lactic acid, and water; The second natural eutectic solvent includes betaine, urea, and water.

2. The method for preparing fish swim bladder collagen peptides according to claim 1, characterized in that, The molar ratio of betaine, lactic acid, and water in the first natural eutectic solvent is 1:(2-4):(1-3); and / or, The molar ratio of betaine, urea and water in the second natural eutectic solvent is 1:(2-5):(1-2).

3. The method for preparing fish swim bladder collagen peptides according to claim 1, characterized in that, The volume ratio of the first natural eutectic solvent to the second natural eutectic solvent is (5-7):(3-5).

4. The method for preparing fish swim bladder collagen peptides according to claim 1, characterized in that, The ratio of the fish swim bladder to the composite natural eutectic solvent is 1:(15-25)(g / mL).

5. The method for preparing fish swim bladder collagen peptides according to any one of claims 1 to 4, characterized in that, The ultrasound power is 500W-700W, and the duration is 5 min-20 min.

6. The method for preparing fish swim bladder collagen peptides according to any one of claims 1 to 4, characterized in that, The complex protease includes two or more of the following: alkaline protease, papain, bromelain, neutral protease, flavor protease, trypsin, and pepsin. Optionally, the conditions for the first step of enzymatic hydrolysis include: the mass of the elastase is 0.5%-2.5% of the mass of the fish swim bladder, the pH is 8.5-9.5, the temperature is 50℃-65℃, and the time is 2h-4h; Optionally, the conditions for the second enzymatic hydrolysis step include: the mass of the complex protease is 1%-4% of the mass of the fish swim bladder, the pH is 7.5-8.5, the temperature is 50℃-60℃, and the time is 3h-5h.

7. The method for preparing fish swim bladder collagen peptides according to claim 6, characterized in that, The method satisfies one or more of the following conditions: (1) The amount of elastase added is 0.6%-0.8% of the mass of the fish swim bladder; (2) The amount of the compound protease added is 1.4%-1.6% of the mass of the fish swim bladder; (3) The complex protease is an alkaline protease, a neutral protease, and a flavor protease; Optionally, the mass ratio of the alkaline protease, the neutral protease, and the flavor protease is 1:(0.5-1):(0.2-0.5).

8. The method for preparing fish swim bladder collagen peptides according to any one of claims 1 to 4, 6 to 7, characterized in that, The enzyme inactivation process also includes separation and purification processes as well as drying processes. Optionally, the separation and purification process includes first filtration with an ultrafiltration membrane with a molecular weight cutoff of 8 kDa-12 kDa, and then second filtration with a nanofiltration membrane with a molecular weight cutoff of 180 Da-220 Da. Optionally, the drying process includes spray drying, and the parameters for spray drying include an outlet air temperature of 85℃-90℃ and an inlet air temperature of 170℃-190℃.

9. A fish swim bladder collagen peptide, characterized in that, It is prepared using the method for preparing fish swim bladder collagen peptides according to any one of claims 1 to 8.

10. The use of the fish swim bladder collagen peptide according to claim 9 in the preparation of products with functions of promoting bone health and resisting skin photodamage.

Images