A pollock bone collagen peptide, a preparation method and application thereof

Abstract

The application discloses narrow hake bone collagen protein peptide and a preparation method and application thereof, and belongs to the technical field of biotechnology. The preparation method of the narrow hake bone collagen protein peptide comprises the following steps: after narrow hake bones are crushed, the narrow hake bones are added with ultrapure water to obtain a mixture, a compound protease composed of alkaline protease and trypsin is added into the mixture to perform enzymolysis, the enzymolysis liquid is filtered through a microporous filter membrane to obtain an enzyme hydrolysate; the enzyme hydrolysate is separated through a 3k Da ultrafiltration membrane, components with a molecular weight less than 3kDa are collected, and after being suction filtered, the narrow hake bone collagen protein peptide is obtained through gel chromatography separation. The narrow hake bones are used as raw materials, three polypeptides with anti-fatigue activity and anti-inflammatory activity are prepared and screened, the polypeptides are artificially synthesized, a narrow hake bone collagen protein anti-fatigue and anti-inflammatory activity peptide chewable tablet product is developed, and reference bases are provided for high-value utilization of aquatic product processing byproduct protein resources.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a pollock bone collagen peptide, its preparation method, and its application. Background Technology

[0002] Alaskan pollock, also known as Alaska pollock or Alaska hockey, belongs to the order Gadiformes, family Gadidae, and genus Alaska. It is highly sought after due to its rich content of high-quality protein, balanced amino acid composition, and advantages such as high protein, low fat, tender flesh, affordable price, and ease of cooking. Current research on pollock focuses primarily on storage, preservation, and processing. For example, extending the shelf life of prepared pollock fillets using natural biological preservatives, inhibiting spoilage of cod meat using edible films, and exploring the impact of salting on the quality of pollock fillets are all being investigated. However, during the primary processing of pollock, over 60% of the raw material generates byproducts including fish heads, skin, bones, minced meat, and viscera. Among these, pollock bones, as a major solid byproduct, are rich in calcium, phosphorus, trace elements, protein, and unsaturated fatty acids, possessing potential benefits such as preventing and improving osteoporosis, enhancing immunity, and maintaining cardiovascular health. However, due to limitations in extraction and processing technologies, most of these byproducts are disposed of in low-value form or discarded directly, resulting in a huge waste of resources and a negative impact on the environment.

[0003] Although gelatin extraction and the preparation of various bioactive peptides, such as antioxidant peptides and metal chelate peptides, have been achieved using cod bones as raw materials, the bioactive peptides developed for Alaska pollock bones currently tend to have relatively singular functions, only meeting one specific need in a particular field. For example, antioxidant peptides are mainly used to delay oxidation reactions, while metal chelate peptides focus on binding metal ions. With the increasing demands for bioactive peptides in the food, pharmaceutical, and cosmetic industries, single-function bioactive peptides are no longer sufficient to meet complex market needs. The industry urgently needs bioactive peptides that possess multiple functional properties simultaneously, such as combined antioxidant and immune-enhancing functions. Collagen peptides, with their excellent bioactivity and functional properties, have shown broad application prospects in many fields. Therefore, developing novel bioactive peptides with comprehensive functions from Alaska pollock bones and deeply exploring their potential functional properties and application value is of vital importance for improving the utilization rate of Alaska pollock bone resources, expanding industrial development space, and creating greater economic and environmental benefits. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing and applying Alaska pollock bone collagen peptides, thereby addressing the problems existing in the prior art. This invention uses Alaska pollock bone as raw material and employs a bio-enzymatic hydrolysis method to prepare collagen peptides. Three polypeptides with both anti-fatigue and anti-inflammatory activities were screened out. These polypeptides were then synthesized artificially using solid-phase synthesis to develop a chewable peptide product containing Alaska pollock bone collagen peptides with anti-fatigue and anti-inflammatory activities. This provides a reference for the high-value utilization of protein resources from aquatic product processing byproducts.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] This invention provides a method for preparing collagen peptides from pollock bone, comprising the following steps:

[0007] Pollock bones were crushed and added to ultrapure water to obtain a mixture. A complex protease consisting of alkaline protease and trypsin was added to the mixture for enzymatic hydrolysis. The mixture was then filtered through a microporous membrane to obtain the hydrolysate.

[0008] The enzymatic hydrolysate was separated by a 3kDa ultrafiltration membrane, and the components with a molecular weight <3kDa were collected. After filtration, the components were separated by gel chromatography to obtain the Alaska pollock bone collagen peptides.

[0009] Furthermore, the ratio of the pollock bone to the ultrapure water is 1g:15.8mL.

[0010] Furthermore, the mass ratio of the alkaline protease to the trypsin is 1:0.5; and the amount of the complex protease added is 0.8% of the mass of the mixture.

[0011] Furthermore, the enzymatic hydrolysis temperature is 50°C and the enzymatic hydrolysis time is 4.5 h.

[0012] Furthermore, the gel chromatography separation used dextran gel G-15 as the packing material, with a sample concentration of 30 mg / mL and a sample volume of 2 mL; the sample was washed with deionized water at a flow rate of 1 mL / min and eluted with ultrapure water.

[0013] The present invention also provides a pollock bone collagen peptide obtained by the preparation method described above.

[0014] Furthermore, the pollock bone collagen peptide comprises any one or more amino acid sequences as shown in SEQ ID NO. 2-4.

[0015] The present invention also provides the application of the aforementioned pollock bone collagen peptide in the preparation of products with anti-inflammatory and anti-fatigue effects.

[0016] The present invention also provides a product with anti-inflammatory and anti-fatigue effects, the active ingredient of which includes the aforementioned pollock bone collagen peptide.

[0017] Furthermore, the product includes chewable tablets.

[0018] The present invention discloses the following technical effects:

[0019] This invention uses Alaska pollock bone as raw material and prepares collagen peptides through enzymatic hydrolysis. After separation and purification by gel chromatography, multiple bioactive peptides are identified using LC-MC / MS technology. Furthermore, three peptides exhibiting both anti-fatigue and anti-inflammatory activities are screened through docking with MAPK1. The anti-fatigue and anti-inflammatory activities of the peptides are further verified through animal and cell experiments. By artificially synthesizing peptides in a solid phase, a chewable peptide product containing Alaska pollock bone collagen with anti-fatigue and anti-inflammatory activity has been developed, providing a reference for the high-value utilization of protein resources from aquatic product processing byproducts. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0021] Figure 1 The molecular weight distribution of the enzymatic hydrolysate of Alaska pollock bones;

[0022] Figure 2 The DPPH removal rate and reducing power of different ultrafiltration components;

[0023] Figure 3 The effect of pollock bone collagen peptides on mouse body weight;

[0024] Figure 4 The effects of pollock bone collagen peptides on the duration of weight-bearing exhaustion swimming (A) and fatigue rotarod duration (B) in mice;

[0025] Figure 5 The effects of pollock bone collagen peptides on serum BUN (A), LA (B), and CK (C) levels, which are fatigue-related indicators, in mice.

[0026] Figure 6 The effects of pollock bone collagen peptides on mouse glycogen LG(A) and MG(B);

[0027] Figure 7 The effects of pollock bone collagen peptides on the levels of MDA (A), SOD (B), GSH-Px (C), and CAT (D) in mouse muscle;

[0028] Figure 8The effect of pollock bone collagen peptides on serum TNF-α (A) and IL-1β (B) levels in mice;

[0029] Figure 9 Gel chromatography separation pattern (A) and DPPH· scavenging rate and reducing power of different components (B);

[0030] Figure 10 The LC-MS / MS mass spectrum of the peptide KGWK;

[0031] Figure 11 The effects of synthetic peptides GPSGIR (A), KGWK (B), and SIFQR (C) on cell viability;

[0032] Figure 12 The effects of synthetic peptides GPSGIR (A), KGWK (B), and SIFQR (C) on TNF-α and IL-1β production in cells were investigated.

[0033] Figure 13 The antioxidant activity of the pollock bone collagen peptide chewable tablets and the control group was evaluated. Detailed Implementation

[0034] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0035] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0036] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0037] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This specification and embodiments are merely exemplary.

[0038] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0039] Example 1: Preparation of Alaska pollock bone collagen peptides

[0040] 1. Preparation of Alaska pollock bone collagen peptides by enzymatic hydrolysis

[0041] A certain mass of fish bone powder was added to ultrapure water at a material-to-liquid ratio of 1:15.8 (g:mL), and the pH was adjusted to 8.2. A complex protease consisting of alkaline protease and trypsin (mass ratio of alkaline protease to trypsin of 1:0.5) was added at 0.8 wt% for enzymatic hydrolysis. The hydrolysis temperature was 50℃, and the hydrolysis time was 4.5 h. After hydrolysis, the enzyme was inactivated by boiling water for 15 min. After cooling, the mixture was centrifuged (8000 r / min) for 15 min at 4℃. The supernatant was filtered through a 0.45 μm microporous membrane to obtain the hydrolysate. The hydrolysate was pre-cooled overnight at -80℃ and then freeze-dried to obtain collagen peptide powder for later use.

[0042] 2. Molecular weight distribution of collagen peptides from Alaska pollock bones

[0043] Molecular weight was determined using high-performance gel permeation chromatography (HPLC). Pollock bone collagen peptide powder was diluted to 1 mg / mL. The detection conditions were as follows: mobile phase: acetonitrile:water:trichloroacetic acid (40:60:0.05); column type: Shodex Asahipak GS-320HQ; flow rate: 0.5 mL / min; detection wavelength: 220 nm. Standard samples included: cytochrome C, aprotinin, bacitracin, acetaminophen-acetaminophen-tyrosine-arginine, and acetaminophen-acetaminophen-acetaminophen. The determination method followed GB-31645-2018.

[0044] Test results as follows Figure 1 As shown, after hydrolysis by protease, the large molecular proteins in the pollock bone were hydrolyzed into small molecular peptides. Among them, the content of peptides with a molecular weight of less than 3 kDa reached 94.81%, and the content of peptides with a molecular weight of less than 0.5 kDa reached 55.83%. This indicates that the content of small molecular peptides obtained after enzymatic hydrolysis of pollock bone is high, the degree of enzymatic hydrolysis is high, and the enzymatic hydrolysis effect is good.

[0045] Example 2: Anti-fatigue effect of Alaska pollock bone collagen peptides

[0046] 1. Ultrafiltration separation of enzymatic hydrolysate from Alaska pollock bones

[0047] Ultrafiltration membranes with a molecular weight less than 3 kDa typically exhibit strong biological activity and are better absorbed by the intestines; membranes with a molecular weight between 3 kDa and 10 kDa generally possess good stability and moderate biological activity. Therefore, this invention uses 3 kDa and 10 kDa ultrafiltration membranes to separate the components of the enzymatic hydrolysate. The specific method is as follows:

[0048] Take the enzymatic hydrolysate of pollock bone prepared in Example 1, select an ultrafiltration membrane with a molecular weight cutoff of 3kDa and 10kDa, and perform ultrafiltration separation at 4°C. Collect three components with molecular weights of <3kDa, 3kDa to 10kDa, and >10kDa, respectively. After freeze-drying, store at -80°C for later use.

[0049] 2. Determination of antioxidant activity

[0050] The DPPH scavenging power and Fe were obtained from the ultrafiltration separation of the three components. 3+ The reducing power was measured, and the results are as follows: Figure 2 As shown, the antioxidant capacity of components with molecular weights <3kDa and >10kDa was significantly improved compared to the untreated enzymatic hydrolysate. However, the antioxidant capacity of components with molecular weights in the range of 3k to 10kDa was significantly reduced compared to the control group. This indicates that molecular weight has a significant impact on the antioxidant activity of the samples. Peptides with strong antioxidant activity may have certain anti-fatigue effects. Considering all factors, collagen peptides with molecular weights <3kDa were selected for subsequent experiments.

[0051] 3. Animal experiments demonstrating anti-fatigue effects

[0052] 3.1 Mouse grouping and administration

[0053] Twenty-four male ICR mice (4 weeks old, SPF grade) were selected and randomly divided into four groups of six mice each after five days of acclimatization. The specific grouping and drug dosage are as follows:

[0054] (1) NC group: 1 mL / 100 g*d of physiological saline was administered by gavage;

[0055] (2) PC group: 0.5 mg / g*d of glutathione was administered by gavage;

[0056] (3) LDG group: Alaska pollock bone collagen peptides were administered by gavage at a dose of 0.25 mg / g*d.

[0057] (4) HDG group: 1 mg / g*d of pollock bone collagen peptide was administered by gavage.

[0058] Housing conditions: 3 mice per cage, room temperature (25±2℃), moderate humidity (50±10%), light / dark cycle of 12 / 12h. Noise level controlled below 60dB.

[0059] Pollock bone collagen peptides were prepared into a solution with ultrapure water before daily administration. All mice were administered the solution by gavage from 9:00 AM to 11:00 AM daily, followed by 30 minutes of swimming training, twice a week. Each mouse was administered the solution at a dose of 1 mL / 100 g for 4 weeks, with free access to water and food throughout the experiment. Initial mouse weight was recorded, and the mice were weighed and their weight (g) recorded weekly during the experiment. On day 28, mice underwent a weight-bearing exhaustion swimming test 30 minutes after gavage. Two hours after the end of the weight-bearing exhaustion swimming test, a fatigue rotarod test was performed.

[0060] Two days after the behavioral experiment, the mice were gavaged for 30 minutes and then swam in a water tank for 30 minutes. After that, the mice were dried and allowed to rest for 30 minutes before blood was collected. Under aseptic conditions, the eyeballs were removed and blood was collected. The blood was placed in a 37°C water bath to prevent coagulation and centrifuged at 4°C and 6000 rpm for 10 minutes. The supernatant serum was aspirated, aliquoted, and stored at -80°C for later use in the determination of relevant biochemical indicators.

[0061] Mice were euthanized by dislocation after blood collection. Under aseptic conditions, the spleen, thymus, kidneys, liver, and hind limb gastrocnemius muscle were removed. Excess adipose tissue was removed, and the organs were rinsed with physiological saline to remove blood from the surface. Excess moisture was absorbed with filter paper, and the organs were aliquoted and stored at -80℃ for later use in subsequent biochemical tests.

[0062] 3.2 Effects of Alaska pollock bone collagen on physiological indicators in mice

[0063] During the 28-day administration period, the weight changes of mice in each group showed roughly the same trend, with no significant difference compared to the NC group, all exhibiting a gradual increasing trend. Figure 3 The results indicate that the mice are growing well and their physiological functions are within the normal range. Furthermore, no abnormal behavior or death occurred in the mice during the experiment. Compared with the NC group, there were no significant differences in organ indices among the intervention groups. This indicates that gavage administration of Alaska pollock bone collagen peptides has no adverse effects on the normal growth, development, and physiological metabolism of mice, and has no toxic side effects, demonstrating its safety.

[0064] 3.3 Effects of Alaska pollock bone collagen on behavioral performance in mice

[0065] The exhaustive swimming test is an ideal experimental model for evaluating the anti-fatigue effect in mice. Improvements in exercise endurance can directly measure the effect of anti-fatigue factors. For example... Figure 4As shown in Figure A, compared with the NC group, the time of weight-bearing exhaustion swimming in all sample treatment groups was significantly increased (P<0.05), with the time being extended by 60.38% and 104.83%, respectively. Among them, the HDG group had the longest time of weight-bearing exhaustion swimming.

[0066] The mouse fatigue rotarod test is used to measure fatigue tolerance in mice and assess overall muscle function. Figure 4 As shown in B, the rotarod time of mice in all sample treatment groups was higher than that in the NC group. Among them, there was no statistically significant difference between the LDG group and the NC group (P>0.05), while there was a significant difference between the HDG group and the NC group (P<0.05), with the time being prolonged by 67.40%, which was better than that of the PC group.

[0067] These results indicate that supplementation with pollock bone collagen peptides can prolong the time of mice swimming at exhaustion under load and the time of fatigue rotarod, and improve the exercise fatigue tolerance of mice.

[0068] 3.4 Effects of Alaska pollock bone collagen on fatigue-related indicators in mice

[0069] The effects of pollock bone collagen peptides on serum BUN (blood urea nitrogen), LA (lactic acid), and CK (creatine kinase) levels in mice are as follows: Figure 5 As shown in the figure, the levels of BUN, LA, and CK in mice in the LDG and HDG groups were lower than those in the NC group. Meanwhile, the levels of the three serum indicators in the LDG group were higher than those in the HDG group, but the differences were not significant compared to the NC group (P>0.05). These results indicate that pollock bone collagen peptides can reduce amino acid metabolism and decrease LA levels in vivo, while also protecting muscles from damage, thereby delaying fatigue.

[0070] 3.5 Effects of Alaska pollock bone collagen on mouse glycogen

[0071] Glycogen is an important indicator for assessing fatigue. For example... Figure 6 As shown, the LG (liver glycogen) levels in mice in the LDG and HDG groups were higher than those in the NC group, but there was no significant difference in LG levels between the LDG and NC groups (P>0.05). The MG (muscle glycogen) levels in mice in the PC and HDG groups were significantly higher than those in the NC group (P<0.05). Compared with the NC group, the LDG group showed a slight increase in MG levels, but the difference was not significant (P>0.05). These results indicate that pollock bone collagen peptides can increase the body's glycogen reserves, regulate blood glucose concentration, provide energy for muscle contraction, and exert an anti-fatigue effect.

[0072] 3.6 Effects of Alaska pollock bone collagen on oxidative stress in mice

[0073] The levels of antioxidant enzymes SOD, GSH-PX, and CAT, as well as lipid peroxidation product MDA, are often used to assess the extent of oxidative damage in the body after exercise.

[0074] The effects of pollock bone collagen peptides on the levels of MDA, SOD, GSH-Px, and CAT in mouse muscle are as follows: Figure 7 As shown. By Figure 7 As shown in A, compared with the NC group, exercise significantly reduced the MDA levels in the muscles of mice in the PC, LDG, and HDG groups by 42.46%, 38.45%, and 36.20%, respectively (P<0.05). Figure 7 B showed that the NC group had the lowest SOD level, while the PC, LDG, and HDG groups showed significantly higher levels (70.25%, 38.47%, and 33.60%, respectively, P<0.05). Observation Figure 7 As shown in C, the GSH-Px levels in the PC, LDG, and HDG groups were all higher than those in the NC group, increasing by 54.98%, 82.14%, and 75.14%, respectively. The LDG group exhibited the highest GSH-Px level at 7.56 ± 0.79 U / mg prot, indicating that Alaska pollock bone collagen peptides were more effective than glutathione. Figure 7 As shown in Figure D, compared with the NC group, the CAT levels in the PC group and HDG group mice increased by 289.64% and 207.48%, respectively. The LDG group had a higher CAT level than the NC group, but the difference was not significant (P>0.05). These results indicate that regular supplementation with pollock bone collagen peptides can improve the antioxidant capacity of mice, prevent lipid oxidation, and thus delay the onset of fatigue.

[0075] 3.7 Effects of Alaska pollock bone collagen on inflammatory factors in mice

[0076] This invention detects TNF-α and IL-1β cytokines in mouse serum. The results are as follows: Figure 8 As shown, after exercise, the HDG group mice showed significantly reduced levels of TNF-α and IL-1β (P<0.05), decreasing by 10.47% and 26.79% respectively compared to the NC group. Although there was no significant difference in levels in the LDG group (P>0.05), it was still lower than that in the NC group. These results indicate that Alaska pollock bone collagen peptides can delay the onset of fatigue by regulating inflammatory factors.

[0077] Example 3: Isolation and purification of collagen peptides from Alaska pollock bone

[0078] 1. Gel chromatography separation of collagen peptides from Alaska pollock bones

[0079] The components with a molecular weight less than 3 kDa obtained by ultrafiltration separation in Example 2 were dissolved in ultrapure water to prepare a solution with a concentration of 30 mg / mL. After being filtered through 0.45 μm and 0.22 μm filter membranes, the solution was then subjected to column chromatography for detection.

[0080] The column conditions were as follows: Sephadex G-15 dextran gel resin; glass column size 3cm × 70cm; sample concentration 30mg / mL; loading volume 2mL; and deionized water rinsing at a flow rate of 1mL / min. Elution was performed with ultrapure water, and the eluent was collected using a fractionating collector, one tube (5mL) every 6 minutes, with absorbance measured at 214nm. The three obtained fractions were collected separately, freeze-dried, and their antioxidant activity was measured. Their DPPH· scavenging rate and Fe2+ scavenging rate were compared. 3+ The reducing power was used to screen out the better components for subsequent experiments.

[0081] Gel chromatography separation patterns as follows Figure 9 As shown in Figure A, dextran gel G-15 can separate the sample into three components, named F-1, F-2, and F-3 respectively. These components were collected, lyophilized, and their antioxidant activities were then determined. The results are as follows: Figure 9 As shown in B, the antioxidant activity of component F-1 was the highest and significantly higher than that of components F-2, F-3 and components with a molecular weight <3kDa (P<0.05). Therefore, component F-1 was selected for the subsequent amino acid sequence identification.

[0082] 2. LC-MS / MS identification of collagen peptides from Alaska pollock bone

[0083] The amino acid sequence of the F-1 fraction separated by gel chromatography was identified using LC-MC / MS. After reductive alkylation, enzymatic digestion, and desalting, 1 μL of sample was loaded. Each sample was analyzed for 60 min at a flow rate of 400 nL / min. The gradient started at 0% in phase B, increased to 28% at 53 min, then to 100% at 4 min, and held for 10 min. Specific mass spectrometry conditions were: full scan range 300–1800 m / z; primary mass spectrometry resolution 60,000, AGC 3e6, maximum injection time 20 ms; secondary mass spectrometry resolution 15,000, AGC 5e4, maximum injection time 22 ms, collision energy 27%. The secondary mass spectrum is shown below. Figure 10 As shown.

[0084] A total of 95 peptide sequences with relatively reliable peptide scores were obtained from F-1. The molecular weight range of F-1 peptides is 300-2000 Da, with a high content of oligopeptides, including 1 18-peptide, 1 16-peptide, 1 14-peptide, 2 13-peptides, 1 12-peptide, 3 11-peptides, 4 10-peptides, 4 9-peptides, 7 8-peptides, 6 7-peptides, 18 6-peptides, 18 5-peptides, 15 4-peptides, 13 3-peptides, and 1 2-peptide.

[0085] 3. Prediction of the physicochemical properties of collagen peptides from Alaska pollock bones

[0086] The bioactivity of Alaska pollock bone collagen peptides was predicted using the online tool Peptide Ranker (https: / / distilldeep.ucd.ie / PeptideRanker / ); the solubility of collagen peptides was predicted using the online tool Innovagen (http: / / www.innovagen.com / proteomics-tools); and the toxicity of collagen peptides was predicted using the online tool ToxinPred (https: / / webs.iiitd.edu.in / raghava / toxinpred / multi_submit.php). Ultimately, 12 peptides with good water solubility, non-toxicity, and high bioactivity were screened for further molecular docking studies.

[0087] 4. Docking results of peptides with MAPK1

[0088] Locate and download the crystal complex structure (ID: 2Y9Q) of human MAPK1 from the RCSB Protein Data Bank using Discovery Studio 2017R. 2 The three-dimensional structures of 12 peptides were constructed, and force fields were applied with energy minimization. These peptides were then docked with the receptor MAPK1 via a CDOCKER semi-flexible molecular docking. The three-dimensional structure of the receptor MAPK1 was optimized using Prepare Protein, followed by dehydration and hydrogenation. The ligand pretreatment parameters were Max Steps-200, RMS gradient-0.1, and Nonbond listradius-14. The molecular docking parameter was Pose Cluster radius-0.5. Other parameters were set to system default values.

[0089] Molecular docking revealed that 5 out of the 12 peptides successfully docked with the receptor MAPK1, and the docking energies are shown in Table 1. As can be seen from the table, peptides GPSGIR (-82.9881 kcal / mol), KGWK (-91.0557 kcal / mol), and SIFQR (-69.7664 kcal / mol) had lower total docking energies, indicating that these three peptides docked more tightly with the receptor MAPK1 and possess potential anti-fatigue activity.

[0090] Table 1. Molecular docking energy results between peptides and receptor MAPK1

[0091]

[0092] Table 2 shows the scores of the binding sites of the pollock bone peptides to the MAPK1 receptor. As can be seen from the table, the main binding sites of the five peptides to the MAPK1 receptor include LYS54, SER153, GLN105, GLU33, LYS114, CYS166, and MET108. Among them, LYS54, SER153, and GLN105 have higher scores, indicating that these three amino acid residues are the most important binding amino acid residues and play a major role in the interaction between pollock bone collagen peptides and the MAPK1 receptor.

[0093] Table 2 Scores of peptide binding sites to receptor MAPK1

[0094]

[0095]

[0096] Note: "+" indicates the number of interactions between the ligand and the receptor.

[0097] 5. Solid-phase synthesis of polypeptides

[0098] The peptides GPSGIR, KGWK, and SIFQR were synthesized by Nanjing Peptide Valley Biotechnology Co., Ltd. The peptides were synthesized in a reactor following a sequence from the sequence to the C-terminus to the N-terminus. Amino acid equivalents were added to the reactor for sequential modification and ligation. After sequence synthesis, a cleavage buffer was added to terminate the reaction. The product was precipitated, centrifuged, and washed to obtain the sequence product. Further peptide purification and lyophilization were then performed, resulting in peptides with a purity exceeding 95%.

[0099] Example 5: Anti-inflammatory effect of Alaska pollock bone collagen peptides

[0100] The synthetic peptides synthesized artificially in solid phase in Example 4 were prepared into complete culture media with concentrations of 0, 50, 100, 200, and 400 μg / mL.

[0101] A lipopolysaccharide (LPS)-induced mouse C2C12 myoblast injury model was established: C2C12 cells were seeded in DMEM medium containing 10% fetal bovine serum (FBS), 1% penicillin / streptomycin, and cultured at 37°C and 5% CO2. When the C2C12 cells reached approximately 80% confluence, they were washed with phosphate-buffered saline (PBS) and cultured again in DMEM medium containing 2% FBS, 1% penicillin / streptomycin, to induce C2C12 cell differentiation for 7 days. Different concentrations of synthetic peptides were added to complete culture medium to pretreat differentiated C2C12 cells for 24 h, followed by stimulation with 0.5 μg / mL LPS solution (dissolved in DMEM) for 6 h. The effects of three Alaska pollock bone synthetic peptides on C2C12 cell survival were determined using the CCK-8 assay. The levels of inflammatory factors in C2C12 cells were measured according to the TNF-α and IL-1β kit instructions.

[0102] The effects of three synthetic peptides on cell survival rate are as follows: Figure 11 As shown in the figure, when the sample concentration was 50-200 μg / mL, all three synthetic peptides significantly promoted cell survival, with KGWK showing the highest survival rate, followed by GPSGIR and SIFQR. However, at a sample concentration of 400 μg / mL, the cell survival rates of the synthetic peptides GPSGIR and SIFQR were inhibited, possibly due to the cytotoxic effects of high concentrations. These results indicate that the synthetic peptides GPSGIR, KGWK, and SIFQR can effectively prevent LPS-induced damage to C2C12 cells, exhibiting a certain anti-inflammatory effect.

[0103] When stimulated by LPS, C2C12 cells release inflammatory factors TNF-α and IL-1β to enhance the body's defense capabilities. The effects of adding three synthetic peptides on the secretion of cytokines IL-1β and TNF-α by LPS-induced C2C12 cells are as follows: Figure 12 As shown in the figure, the levels of IL-1β and TNF-α in the LPS-treated damaged group were significantly higher than those in the normal group (P<5). When synthetic peptides of 50–400 μg / mL were added to the cell model, all concentrations of synthetic peptides could slow down the release of inflammatory factors, and the inhibitory effect was more obvious with increasing concentration. The synthetic peptide KGWK could effectively slow down the release of IL-1β and TNF-α, and had the best inhibitory effect among the three synthetic peptides. At a concentration of 400 μg / mL, the inhibition rate reached 28.86% and 30.55%, respectively. However, high concentrations of GPSGIR had a certain toxic effect on cells, which is similar to the experimental results above.

[0104] Example 6: Alaska pollock bone collagen peptide chewable tablets

[0105] 1. Preparation of Alaska pollock bone collagen peptide chewable tablets

[0106] The components with a molecular weight of less than 3 kDa obtained by ultrafiltration separation in Example 2 were freeze-dried and used as the base material: Alaska pollock bone collagen peptides.

[0107] The base material, pollock bone collagen peptides, flavoring agent (xylitol), milk powder, citric acid, binder (sodium carboxymethyl cellulose), and filler (microcrystalline cellulose) are ground and pulverized separately. After passing through a 100-mesh sieve, water is slowly added and mixed until the material reaches a state where it "clumps together when squeezed but crumbles when touched." The material is then extruded through a 20-mesh standard sieve and dried at a constant temperature of 60℃ for 1 hour (turning over every 0.5 hours) until the moisture content is about 3%. The material is then sieved again through a 20-mesh standard sieve. Lubricant (magnesium stearate) is added to the particles that pass through the sieve, and the mixture is mixed. The tablets are then compressed using a tablet press at a pressure of 0.25–0.30 MPa. After sterilization by ultraviolet irradiation for 1 hour (turning over every 0.5 hours), the finished chewable tablets are obtained. The chewable tablets contain, by weight percentage, 30% pollock bone collagen peptides, 9% sodium carboxymethyl cellulose, 3% citric acid, 15% milk powder, 1% magnesium stearate, 20% xylitol, and 12% microcrystalline cellulose.

[0108] The prepared chewable tablets had a diameter of 12 mm, a thickness of 5 mm, and an average tablet weight of 0.4974 g. The difference in tablet weight between tablets was within 5%.

[0109] 2. Performance Analysis of Chewable Tablets

[0110] 2.1 Determination of texture and disintegration time

[0111] The textural properties of the prepared chewable tablets were determined, with commercially available milk tablets used as a control. The results are shown in Table 3. The Alaska pollock bone collagen peptide chewable tablets were intact in shape, with a smooth and flat surface, no powdery texture, and suitable hardness. Table 3 shows that the hardness of the Alaska pollock bone collagen peptide chewable tablets was lower than that of the commercially available milk tablets, but the difference was not significant. Simultaneously, the differences in viscosity and resilience between the two types of chewable tablets were minimal, and the chewability of the Alaska pollock bone collagen peptide chewable tablets was slightly lower than that of the commercially available milk tablets, indicating that the collagen chewable tablets had better chewability, and their overall textural properties were similar to those of the commercially available milk tablets.

[0112] The disintegration time of the chewable tablets was measured to be (25±0.5) min, which meets the relevant requirement of less than 30 min in the Chinese Pharmacopoeia (2020 edition).

[0113] Table 3. Texture properties of chewable tablets

[0114]

[0115] 2.2 Determination of whiteness value

[0116] A blank group of chewable tablets was prepared by replacing the Alaska pollock bone collagen peptides with fillers, while keeping other dosages unchanged, and the whiteness value of each group was measured.

[0117] The whiteness values ​​of the Alaska pollock bone collagen peptide chewable tablets and the control group are shown in Table 4. The chewable tablets are slightly yellow, while the control group is white. The a* and b* values ​​of the chewable tablets with added Alaska pollock bone collagen peptides are significantly higher than those of the control group. Compared with the control group, the chewable tablets are more yellow and reddish, which is due to the yellowish color value of the collagen peptides themselves.

[0118] Table 4. Whiteness values ​​of chewable tablets

[0119]

[0120] 2.3 Determination of Microbiological Indicators

[0121] The microbiological indicators of the Alaska pollock bone collagen peptide chewable tablets are shown in Table 5. The total bacterial count in the chewable tablets was <100 CFU / g, and no Escherichia coli was detected, which meets the requirements.

[0122] Table 5 Results of Microbiological Indicators Determination of Chewable Tablets

[0123]

[0124] 2.4 Antioxidant Activity of Chewable Tablets

[0125] Antioxidant activity of pollock bone collagen peptide chewable tablets and control group, such as Figure 13 As shown. The DPPH clearance rate of the Alaska pollock bone collagen peptide chewable tablets was 62.71%, which is about 18 times higher than the clearance rate of 3.35% in the blank group with the same mass concentration. Furthermore, the Fe... 3+ The reducing power was 42.38%, which was significantly higher than the 1.97% of the blank group, indicating that the pollock bone collagen peptide chewable tablets prepared in this invention have strong antioxidant activity.

[0126] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A pollock bone collagen peptide, characterized by, The amino acid sequence of the said pollock bone collagen peptide is shown as SEQ ID NO. 2 or SEQ ID NO.

4.

2. Use of the pollock bone collagen peptide of claim 1 in the preparation of an anti-inflammatory product.

3. An anti-inflammatory product, characterized in that, The active ingredient comprises the pollock bone collagen peptide of claim 1.

4. The product of claim 3, wherein, The product comprises chewable tablets.

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