Salmon by-product peptide with anti-freezing effect, anti-freezing agent and application

Abstract

The application discloses a salmon by-product peptide with anti-freezing effect, an anti-freezing agent and application, and belongs to the technical field of bioactive peptides. The amino acid sequence of the salmon by-product peptide is one of SEQ ID NO:1-SEQ ID NO:3. The salmon by-product peptide is prepared by using salmon head, fish bone, fish skin and other processing by-products as raw materials, through enzymolysis, grading and purification, structure identification and activity verification, and is successfully obtained, so that the salmon by-product peptide with excellent anti-freezing effect is obtained, and high-value utilization of aquatic processing by-products is realized. In-vitro anti-freezing activity verification shows that the single peptide segment and the complex peptide segment of the three isolated salmon by-product peptides have good anti-freezing effect. The application perfects the development system of functional active substances of salmon by-products, provides important theoretical basis and technical support for research and development of fish aquatic by-product resource utilization and natural anti-freezing functional additives, and has wide industrial application prospect.

Description

Technical Field

[0001] This invention belongs to the field of bioactive peptide technology, specifically relating to a salmon by-product peptide with antifreeze properties, an antifreeze agent, and its applications. Background Technology

[0002] Salmon processing generates a large amount of byproducts, including heads, bones, and skin, accounting for approximately 30% to 50% of its total weight. These byproducts are rich in high-quality protein, polyunsaturated fatty acids, and other bioactive components, possessing significant potential for resource utilization. However, current methods for processing salmon byproducts primarily focus on low-value utilization, such as direct processing into fishmeal or animal feed, or even disposal as waste. This not only wastes resources but may also cause environmental problems. Therefore, how to achieve high-value utilization of salmon byproducts has become a research hotspot in the fields of food science and bioresources.

[0003] During the frozen storage and transportation of food, the formation and growth of ice crystals are the main causes of protein denaturation, cell structure damage, and quality deterioration. Traditional cryoprotectants such as sugars and polyols, while effective to some extent, may lead to adverse effects such as high calorie content and residual sweetness. Naturally derived antifreeze proteins (AFPs) and their derived peptides have attracted widespread attention in recent years due to their ability to inhibit ice crystal recrystallization and protect cell membranes and protein structures. However, the high extraction cost and limited availability of natural antifreeze proteins restrict their widespread application in actual production. Therefore, the development of efficient, safe, and widely available antifreeze peptides has significant application value.

[0004] Therefore, isolating and screening small molecule peptides with significant antifreeze activity from salmon by-products is of great significance for increasing the added value of salmon by-products and developing new natural cryoprotectants. Summary of the Invention

[0005] In view of the problems existing in the prior art, the purpose of this invention is to provide a salmon by-product peptide with antifreeze effect, an antifreeze agent and its application.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: A salmon by-product peptide with antifreeze properties, wherein the amino acid sequence of the salmon by-product peptide is one of SEQ ID NO:1 to SEQ ID NO:3.

[0007] The above-mentioned salmon by-product peptides with antifreeze properties are used in the preparation of products with antifreeze properties.

[0008] An antifreeze agent comprising at least one peptide segment with an amino acid sequence as shown in SEQ ID NO:1 to SEQ ID NO:3.

[0009] Based on the above scheme, it further includes acceptable carriers or excipients.

[0010] Based on the above scheme, the concentration of the peptide is 2 mg / mL.

[0011] The above-mentioned cryoprotectants are used to protect cells, tissues, food, or pharmaceuticals from damage during freezing.

[0012] Advantages of the technical solution of this invention This invention utilizes salmon by-products such as salmon heads, bones, and skin as raw materials. Addressing the industry's current challenges of low utilization rates and significant resource waste of aquatic by-products, as well as the inadequacy of existing antifreeze products and the scarcity of natural antifreeze substances, this invention successfully isolates salmon by-product peptides with excellent antifreeze effects through enzymatic hydrolysis, fractional purification, structural identification, and activity verification. This achieves high-value utilization of aquatic processing by-products. In vitro antifreeze activity verification shows that among the three isolated salmon by-product peptides, the single peptide SRGF exhibits the best antifreeze protection effect, with a catalase residual activity of 79.50%. The combination of different peptides in pairs demonstrates a significant synergistic effect, with the MRPWM+SRGF combination showing the best antifreeze effect and a catalase residual activity as high as 84.37%.

[0013] This invention features a simple, green, and mild preparation process with widely available and inexpensive raw materials. The resulting peptides are natural, safe, and biocompatible, effectively overcoming the limitations of synthetic antifreeze agents. They are suitable for various applications, including food preservation, cold chain processing, and low-temperature storage of biological agents. This invention also improves the development system for functional active substances from salmon by-products, providing important theoretical basis and technical support for the resource utilization of fish aquatic by-products and the research and development of natural antifreeze functional additives, with broad prospects for industrial application. Attached Figure Description

[0014] Figure 1 Results of residual catalase activity of different ultrafiltration components; Figure 2 Chromatogram of <3kDa peptide fraction separated by Sephadex G-15 gel column; Figure 3 Residual catalase activity of each purified component; Figure 4 The total ion chromatogram is shown in LC-MS / MS. Figure 5 This is a secondary structure diagram of three peptide segments; Figure 6 This is the mass spectrum of the MRPWM peptide. Figure 7 This is the mass spectrum of the EHFMF peptide. Figure 8 This is the mass spectrum of the SRGF peptide. Figure 9 Degree of hydrolysis of salmon byproducts hydrolyzed by different proteases; Figure 10 Residual catalase activity of salmon by-products hydrolyzed by different proteases; Figure 11 Results of residual catalase activity in single peptide solutions; Figure 12 Results of residual catalase activity in complex peptide solutions.

[0015] In the above figures, different letters indicate significant differences (p < 0.05). Detailed Implementation

[0016] The terminology used in this invention, unless otherwise specified, generally has the meanings commonly understood by those skilled in the art. The invention is further described in detail below with reference to specific embodiments and data. The following embodiments are merely illustrative and are not intended to limit the scope of the invention in any way.

[0017] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the experimental materials, reagents, and chemicals used in the following embodiments can be obtained through general channels.

[0018] In the following embodiments, 1. Calculation method of degree of hydrolysis During protein hydrolysis, the pH of the solution drops sharply as protons are released. Adding 0.01M NaOH solution helps maintain the pH. The degree of hydrolysis (DH) can be calculated from the volume of alkali added. Calculation formula: C -- Concentration of NaOH solution, in mol / L; V -- Volume of NaOH solution consumed, mL; The dissociation of α-amino groups; Where pH is the pH value of the hydrolysis system, and pKa is the pKa of the amino group. , where T is the Kelvin temperature; m -- Mass of salmon by-product protein in solution, g; h tot --Total number of peptide bonds in the substrate protein, mmol / g, for salmon by-product protein hot=8.6 (calculated based on the amino acid composition of salmon by-product protein).

[0019] 2. Evaluation of in vitro antifreeze activity Cryoprotective activity of catalase: An equal volume of 2 mg / mL sample solution and 1 mg / mL catalase solution were mixed. The mixture was first frozen at -24°C for 24 h, then thawed at 25°C, and then frozen again at -24°C for 6 h. This process was repeated four times. Catalase activity was measured before and after the freeze-thaw cycles, and expressed as residual catalase activity.

[0020] Catalase activity assay: Add 1.9 mL of distilled water, 0.1 mL of catalase solution and 1 mL of 1% hydrogen peroxide dilution to a quartz cuvette, cover and shake immediately, measure the initial absorbance at a wavelength of 240 nm, and then record the absorbance at 1 min intervals for a total of 4 min. One unit of enzyme activity (U) is defined as a decrease of 0.1 A within 1 min.

[0021] Formula for calculating catalase activity: Catalase activity (%) = [(△ 240 [(×dilution factor×total reaction volume) / (reaction time×total sample volume)]×100 Formula for calculating residual catalase activity: Catalase residual activity (%) = (enzyme activity after freeze-thaw / enzyme activity before freeze-thaw) × 100 In the following examples, the salmon by-products were sourced from the Chengyang Aquatic Products Wholesale Market in Qingdao, Shandong Province.

[0022] Example 1

[0023] The method for extracting bioactive peptides from salmon by-products includes the following steps: (1) Salmon by-products (fish head, fish bones and fish skin) are used as raw materials, minced into no obvious lumps, and prepared into a 10% salmon by-product solution, which is then heated at 90°C for 10 min for pre-denaturation (passivation).

[0024] (2) Add 3000 U / g of acidic protease (Ap) to the above salmon by-product solution and hydrolyze it at pH 3 and 50℃ for 3 h. Stir the protein solution continuously and maintain the pH of the system constant with 0.1M NaOH solution (always maintain the optimal pH). After the enzymatic hydrolysis is completed, place the hydrolysate in a 90℃ water bath for 15 min to inactivate the enzyme. After cooling, centrifuge it in a high-speed refrigerated centrifuge at 4000 r / min for 20 min and control the temperature at 4℃. Filter the supernatant and freeze-dry it to obtain crude salmon by-product peptide powder.

[0025] (3) Dilute the supernatant of the enzymatically hydrolyzed salmon by-product antifreeze peptides, filter it first through a 0.45 μm filter membrane, then through a 0.22 μm filter membrane, and pour it into a container. Use membranes with molecular weights of 10 kDa and 3 kDa for ultrafiltration, with a centrifugal force of 4000 r / min, an operating temperature of 25 °C, and times of 15 min and 40 min, respectively. After ultrafiltration, freeze-dry the filtrate to obtain salmon by-product active peptide powders with molecular weights >10 kDa, 3-10 kDa, and <3 kDa.

[0026] Sample peptide solutions of 2 mg / mL were prepared, and the antifreeze activity of salmon by-product bioactive peptides with molecular weights >10 kDa, 3-10 kDa, and <3 kDa was determined. The results are as follows: Figure 1 As shown, there are significant differences in antifreeze activity among components with different molecular weights. Among them, the <3kDa component exhibited the strongest antifreeze activity, with a residual catalase activity of 71.11%.

[0027] (4) The ultrafiltration fraction <3kDa was further purified by filtration on a Superdex™ Increase 10 / 300GL gel permeation column (10×300mm). The column was eluted with deionized water (pH 10), and the fraction was collected at a flow rate of 1 mL / min. The fraction was detected at 280 nm. Four peptide fractions were collected in the order of peak elution and named F1, F2, F3, and F4, respectively. Figure 2 ).

[0028] The residual catalase activities of the above four polypeptide components related to antifreeze activity were measured, and the results are as follows: Figure 3 As shown, there were significant differences in the inhibitory activities of different components (p<0.05): the F2 component had the highest residual catalase activity, reaching 73.33%, which was significantly higher than other components (p<0.05). This result indicates that under these separation conditions, the medium molecular weight F2 component is the main enriched part of highly active antifreeze peptides.

[0029] (5) The active peptides of the F2 fraction with the most antifreeze activity obtained above were screened and analyzed by liquid chromatography-mass spectrometry (LC-MS) to identify the structure of the active peptides. The salmon by-product peptide peaks of the F2 fraction with the most antifreeze activity were separated by an RSLC C18 column using an Easy-nLC high performance liquid chromatography system. Gradient elution: mobile phase A: 0.1% formic acid; mobile phase B: 0.1% formic acid, 80% acetonitrile; the mass spectrometry scanning mode was information-dependent acquisition mode, the primary mass spectrometry scanning resolution was 70000, and a maximum of 20 secondary spectra with charges of 2+ to 5+ were acquired in each working cycle.

[0030] The LC-MS / MS total ion chromatogram of antifreeze peptides from salmon by-products is shown below. Figure 4 As shown, a total of 1400 peptides were obtained by liquid chromatography-mass spectrometry (LC-MS / MS).

[0031] (6) The above 1400 peptides were screened using bioinformatics technology (Table 1). Peptides with MASS molecular weight <1000, no PTM modification, ALC sequence confidence >50%, and Area peak area <10 were selected. 7 The potential bioactivity of the obtained salmon by-product peptides was predicted using the Peptide Ranker program (0-1). Peptides with scores >0.8 were selected, and peptides with predicted toxicity thresholds <0.2 were selected using the ToxinPred 3.0 website. After screening, three peptides (MRPWM, EHFMF, and SRGF) were finally obtained. The secondary structures of the three peptides are as follows: Figure 5 As shown.

[0032] Table 1. Overview of Peptide Screening (7) The three peptides MRPWM, EHFMF, and SRGF were chemically synthesized. The mass spectra of the three peptides are shown below. Figures 6-8 As shown.

[0033] Example 2

[0034] Effects of protease type on the degree of hydrolysis and antifreeze activity of salmon by-products A 10% salmon by-product solution was prepared and pre-denatured (passivated) by heating at 90℃ for 10 min. Then, trypsin, alkaline protease, neutral protease, flavor protease, pepsin, and papain were added at 3000 U / g each. The optimal conditions for the protease reaction were used as the temperature and pH of the hydrolysis system during the enzymatic hydrolysis process (Table 2). The protein solution was continuously stirred, and the pH of the system was maintained constant using 0.1M NaOH solution (always maintained at the optimal pH). Hydrolysis was carried out for 3 h. The hydrolysate was then placed in a 90℃ water bath for 15 min to inactivate the enzymes. After cooling, it was centrifuged in a high-speed refrigerated centrifuge at 4000 r / min for 20 min, with the temperature controlled at 4℃. The supernatant was filtered and freeze-dried to obtain crude salmon by-product peptide powder.

[0035] Table 2. Optimal temperature and pH for different proteases The effects of different proteases on the degree of hydrolysis (DH, %) and in vitro antifreeze activity of salmon by-products were determined, and the results are as follows: Figure 9 and Figure 10 As shown, the salmon by-products hydrolyzed using acidic protease (Ap) exhibited the highest degree of hydrolysis, at 12.76%. Figure 9The cryoprotective capabilities of different enzymatic hydrolysates varied significantly (p<0.05). The acidic protease (Ap) hydrolysate showed the best protective effect, with a residual catalase activity of 66.17%, significantly higher than other treatment groups. In contrast, the pepsin (Pe) and alkaline protease (Al) hydrolysates exhibited relatively weaker protective effects. This result indicates that acidic protease hydrolysates can effectively maintain the activity stability of catalase during repeated freeze-thaw cycles, demonstrating excellent cryoprotective potential.

[0036] Example 3

[0037] A salmon by-product peptide with antifreeze properties, wherein the amino acid sequence of the salmon by-product peptide is shown in SEQ ID NO:1~SEQ ID NO:3.

[0038] SEQ ID NO:1: MRPWM; SEQ ID NO:2: EHFMF; SEQ ID NO:3: SRGF.

[0039] Example 4

[0040] A bioactive peptide preparation with antifreeze properties, wherein the active ingredient is at least one peptide segment of the amino acid sequence shown in SEQ ID NO:1 to SEQ ID NO:3.

[0041] SEQ ID NO:1: MRPWM; SEQ ID NO:2: EHFMF; SEQ ID NO:3: SRGF.

[0042] The three peptides were each prepared into 2 mg / mL solutions using ultrapure water. The effects of the three synthetic peptides on the cryoprotection of catalase were determined, and the results are as follows: Figure 11 As shown, among the single peptides, SRGF exhibited the best catalase residual activity, with an inhibition rate of 79.50%, which was significantly higher than that of MRPWM and EHFMF.

[0043] The synthesized peptides were combined in pairs, and the antifreeze activity of the resulting MRPWM+EHFMF, MRPWM+SRGF, and EHFMF+SRGF composite peptide solutions (2 mg / mL) was verified. The effects of the composite peptide solutions on the cryoprotective activity of catalase were as follows: Figure 12As shown, the overall activity of the complex peptides is superior, with the MRPWM+SRGF combination exhibiting the highest inhibition rate at 84.37%, demonstrating a significant synergistic effect. This complex peptide, as a natural cryoprotectant, combines high activity with safety, providing core component support for the high-value development of antifreeze functional products from salmon by-products.

[0044] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention in any other way. Any person skilled in the art may make changes or modifications to the above-disclosed technical content to create equivalent embodiments. However, any simple modifications, equivalent changes, and modifications made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A salmon by-product peptide with antifreeze properties, characterized in that, The amino acid sequence of the salmon by-product peptide is one of SEQ ID NO:1 to SEQ ID NO:

3.

2. The use of the salmon by-product peptide with antifreeze properties as claimed in claim 1 in the preparation of products with antifreeze properties.

3. An antifreeze agent, characterized in that, It contains at least one of the peptides with amino acid sequences as shown in SEQ ID NO:1 to SEQ ID NO:

3.

4. The antifreeze agent according to claim 3, characterized in that, It may further include acceptable carriers or excipients.

5. The antifreeze agent according to claim 3, characterized in that, The concentration of the peptide is 2 mg / mL.

6. The use of the antifreeze agent according to any one of claims 3-5 in protecting cells, tissues, food or pharmaceuticals from damage during freezing.

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