Tilapia mossambica scale collagen peptide as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of biological polypeptide preparation, and particularly relates to a tilapia scale collagen peptide as well as a preparation method and application thereof. The tilapia mossambica scale collagen peptide is composed of the following peptide fragments: PDPFRMY, GPQGPIGPR, PFRMY, GPSGPP, APDPFR, KAPDPFRMY, PGPGPMGL, DPGPGPMG, DPFRMY, PGGLP, PGPGMP, GPFS, FGAG, PLGP, GPLG, PGR, GPAGGL and PGGGF. The tilapia mossambica scale collagen peptide prepared by the invention has no toxicity, can improve the phagocytic ability of RAW264.7 cells and promote the cells to secrete NO, and can effectively enhance the immunocompetence; the screened tilapia mossambica scale collagen peptide is easier to digest and absorb and higher in bioavailability; the composition can participate in development and migration of cells and tissues and formation of extracellular matrixes, and promotes regeneration of bones, skin and cartilages.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological polypeptide preparation, and particularly relates to a tilapia scale collagen peptide and a preparation method and application thereof. Background Art

[0002] Tilapia is a key farmed fish in the global aquaculture industry and a competitive "key product" for Chinese agricultural exports. Tilapia is primarily processed for frozen and sashimi processing. Tilapia processing byproducts, such as scales, are rich in collagen. Extracting this collagen can create high-value-added uses for tilapia. Tilapia scales, excluding water, are composed of approximately 50% protein, 40% ash, and trace amounts of fat, similar to bone. The proteins in tilapia scales are primarily collagen and sclerostin, with collagen comprising 11.59% to 38.30% of the dry matter.

[0003] The enzyme commonly used in the enzyme extraction method is pepsin. Pepsin acts on the terminal peptides to cut the cross-links between the collagen proteins. The extracted collagen is called pepsin-soluble collagen. Enzyme extraction is green, safe, has few by-products, and does not contain residual organic solvents, but the yield is low. The principle of acid extraction of collagen is that hydrogen ions are conducive to the entry of water into collagen fibers, causing the collagen fibers to swell and dissolve. The protein extracted by acid is called acid-soluble collagen. Acid extraction is time-consuming, produces wastewater, and subsequent neutralization causes the protein salt content to be high. In the prior art, there is no record of tilapia scale collagen peptides promoting the proliferation of RAW264.7 and thus increasing the secretion of NO to enhance the immune system. Summary of the Invention

[0004] In view of the problems existing in the prior art, the present invention provides a tilapia scale collagen peptide.

[0005] The present invention also provides a method for preparing the tilapia scale collagen peptide.

[0006] Another object of the present invention is to provide an application of the tilapia scale collagen peptide.

[0007] The technical solutions adopted by the present invention to achieve the above-mentioned purpose are as follows: The present invention provides a tilapia scale collagen peptide, which is composed of the following peptide segments: PDPFRMY (SEQ ID NO.1), GPQGPIGPR (SEQ ID NO.2), PFRMY (SEQ ID NO.3), GPSGPP (SEQ ID NO.4), APDPFR (SEQ ID NO.5), KAPDPFRMY (SEQ ID NO.6), PGPGPMGL (SEQ ID NO.7), DPGPGPMG (SEQ ID NO.8), DPFRMY (SEQ ID NO.9), PGGLP (SEQ ID NO.10), PGPGMP (SEQ ID NO.11), GPFS (SEQID NO.12), FGAG (SEQ ID NO.13), PLGP (SEQ ID NO.14), GPLG (SEQ ID NO.15), PGR, GPAGGL (SEQ ID NO.16), and PGGGF (SEQ ID NO.17).

[0008] The present invention also provides a method for preparing the tilapia scale collagen peptide, comprising the following steps: (1) Soaking tilapia scales in a citric acid solution, ultrasonically treating the scales, cleaning the scales until neutral, filtering, and drying to obtain de-ashed scales; (2) Mixing dried de-limed fish scales with deionized water, heating the mixture, filtering and concentrating it to obtain gelatin; (3) Add gelatin to pure water, mix well, add enzyme, and perform enzymatic hydrolysis. After the enzymatic hydrolysis is completed, inactivate the enzyme, cool to room temperature, and centrifuge to collect the supernatant to screen tilapia scale collagen peptides.

[0009] Preferably, in step (1), the ratio of the tilapia scales to the 4% citric acid solution is 1:10 (W / V); and the ultrasonic treatment is performed at a power of 40 W for 90 min.

[0010] Preferably, in step (2), the ratio of the delimed fish scales to deionized water is 1:15 (W:V); the pH is adjusted to 5.5; and the heating is performed at 80° C. for 2 h.

[0011] Preferably, in step (3), the material-liquid ratio of gelatin to water is 1:20-100; and the amount of enzyme added is 1000-12000 U / g.

[0012] Preferably, in step (3), the enzyme is alkaline protease, papain, trypsin, pepsin or neutral protease.

[0013] Preferably, in step (3), the enzymatic hydrolysis is carried out at 35-45°C and pH 3-10 for 4.5 hours.

[0014] Preferably, the tilapia scale collagen peptides are screened from enzymatic hydrolysates with a PeptideRanker score of >0.8 and a molecular weight of <3 kDa.

[0015] The present invention also provides the use of the tilapia scale collagen peptide in preparing products for enhancing immune activity.

[0016] The beneficial effects of the present invention are: (1) The extraction method provided by the present invention accelerates molecular vibrations due to heat, disrupting hydrophobic interactions and hydrogen bonds due to changes in molecular arrangement, thereby promoting collagen dissolution and achieving a high extraction rate. By selecting appropriate enzymes, the enzymatic hydrolysis product is standardized, eliminating the need for pH neutralization, resulting in relatively low salt production, mild enzymatic hydrolysis conditions, and preventing amino acid degradation. Using a single-factor response surface optimization approach to enzymatic hydrolysis of collagen, a collagen hydrolyzate with high protein yield and high immune-enhancing activity was obtained.

[0017] (2) The tilapia scale collagen peptide prepared by the present invention is non-toxic and can improve the phagocytic ability of RAW264.7 cells and promote the secretion of NO by cells, which can effectively enhance immune activity; and the tilapia scale collagen peptide screened by the present invention is easier to digest and absorb, and has higher bioavailability; it can participate in the development and migration of cells and tissues and the formation of extracellular matrix, and promote the regeneration of bones, skin, and cartilage. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is the yield of tilapia scale collagen peptides hydrolyzed by different proteases; Figure 2 Effects of tilapia scale collagen peptides hydrolyzed by different proteases on NO secretion in RAW264.7 cells; Figure 3 Effects of different time (a), enzyme dosage (b), temperature (c), and solid-liquid ratio (d) on peptide yield and promotion of NO secretion in RAW264.7 cells; Figure 4 Contour plots and response surface plots under the interaction between enzyme dosage and material-liquid ratio; Figure 5 Contour plots and response surface plots for the interaction between enzyme dosage and temperature; Figure 6 Contour plots and response surface plots under the interaction between solid-liquid ratio and temperature; Figure 7 Effects of <3kDa tilapia scale collagen peptide on RAW264.7 cell proliferation; Figure 8 Effects of <3kDa tilapia scale collagen peptide on the phagocytic ability of RAW264.7 cells; Figure 9 Effects of <3kDa tilapia scale collagen peptide on NO secretion in RAW264.7 cells; Figure 10 Effects of <3kDa tilapia scale collagen peptide on RAW264.7 cell morphology; Figure 11 FTIR spectrum of <3kDa tilapia scale collagen peptide; Figure 12 Molecular weight distribution of <3kDa tilapia scale collagen peptides; Figure 13 The data of peptide segments PDPFRMY, GPQGPIGPR, and GPSGPP on CCK-8 proliferation activity of RAW264.7 cells. DETAILED DESCRIPTION

[0019] The technical solution of the present invention is further explained and illustrated by means of specific embodiments below.

[0020] Example 1 1.1 Tilapia scale collagen extraction Soak the fish scales in a 4% citric acid solution at a material-to-liquid ratio (W:V) of 1:10 and treat with ultrasonic waves at 40W for 90 minutes. Wash the fish scales until neutral, filter with filter cloth, and dry at 50°C to obtain delimed fish scales.

[0021] Dried delimed fish scales and deionized water were mixed in a ratio of 1:15 (w:v) and a pH of 5.5. The mixture was heated at 80°C for 2 hours and then filtered and concentrated (rotary evaporation at 60°C) until the volume was reduced to one-third of its original volume. This gelatin was obtained by recording the volume of the gelatin, measuring its protein concentration using BCA, and calculating the protein extraction yield.

[0022] 1.2 Extraction of fish scale polypeptides Approximately 0.2g of collagen extracted from fish scales was added to purified water at a 2% (g / ml) material-to-liquid ratio. Enzyme was added at 6000 IU / g. Hydrolysis was performed at the enzyme's optimal temperature and pH, with sodium hydroxide and hydrochloric acid used to adjust the pH. After the hydrolysis was complete, the sample was heated in boiling water for 15 minutes to inactivate the enzyme. After cooling to room temperature, the sample was centrifuged at 7000 rpm for 10 minutes, and the supernatant was collected.

[0023] 1.3 Determination of polypeptide yield At 20, 40, 60, 90, 120, 150, 180, and 270 minutes, 500 μL of the enzymatic hydrolyzate was collected and heated in boiling water for 15 minutes to inactivate the enzyme. Then, 500 μL of 5% trichloroacetic acid was added and mixed to precipitate the macromolecular proteins. The mixture was allowed to stand for 10 minutes. After balancing, the solution was centrifuged at 7000 rpm for 15 minutes at 4°C. The supernatant was collected and 1 or 2 μL of the supernatant at each time point was diluted to 20 μL with PBS. The peptide content was measured using a BCA assay. Under alkaline conditions, the peptide bonds in the protein react with Cu2+ to generate Cu+. The Cu+ binds to the BCA reagent, forming a stable purple complex with a characteristic absorption peak at 562 nm. The color intensity is proportional to the protein concentration. The peptide yield was calculated using the formula.

[0024] Peptide yield (%) = C × f × V / m × 100% Where: C——enzyme hydrolysate concentration, mg / ml; F——dilution factor; V——total volume of enzymatic hydrolysate, ml; M——Collagen mass, mg.

[0025] 1.4 Determination of NO secretion Measure the protein concentration of the enzymatic hydrolysate at the end of the enzymatic hydrolysis. Dilute the supernatant obtained in 1.2 10 times and 100 times with culture medium for cell experiments. RAW264.7 cells were used with 2X10 5 Plate the cells at a concentration of 100 μl per well and incubate for 24 hours. Remove the supernatant and add 100 μl of the diluted sample solution. Incubate for 24 hours. Analyze the effects of the sample on NO secretion in RAW264.7 cells using a NO assay kit.

[0026] 1.5 Screening of proteases Five proteases (alkaline protease, papain, trypsin, pepsin, and neutral protease) were added at a ratio of 6000 U / g. Reactions were performed at their respective optimal temperature and pH conditions, using the same 2% substrate concentration. Peptide yields were measured at different time points, and the final enzymatic hydrolysis solution was collected and assayed for NO secretion in RAW264.7 macrophages.

[0027] 1.6 Single-factor experimental design Through the above selection of proteases, the peptide yield and the concentration of NO promoted by RAW264.7 cells were used as indicators of enzymatic hydrolysis effect, and the effects of time, solid-liquid ratio, enzyme dosage and temperature on the enzymatic hydrolysis effect were studied respectively.

[0028] (1) Effect of enzymatic hydrolysis time The enzymatic hydrolysis time was set to 10 min, 20 min, 60 min, 120 min, and 180 min, and the other factors were set as: substrate mass fraction 2%, enzymatic hydrolysis temperature 37°C, enzyme dosage 6000 U / g, and pH 3. The peptide yields of the enzymatic hydrolysates with different enzymatic hydrolysis times and their effects on NO secretion by RAW264.7 cells were compared to determine the optimal enzymatic hydrolysis time.

[0029] (2) Effect of enzyme dosage The enzyme dosage was set at 1000, 3000, 6000, 9000 U / g, and 12000 U / g, and the other factors were set as: substrate mass fraction 2%, enzymatic hydrolysis time 1 h, enzymatic hydrolysis temperature 37°C, and pH 3. The peptide yields and effects of the enzymatic hydrolysis solutions with different enzyme dosages on NO secretion by RAW264.7 cells were compared to determine the optimal enzyme dosage for enzymatic hydrolysis.

[0030] (3) Effect of enzymatic hydrolysis temperature The enzymatic hydrolysis temperatures were set at 30°C, 35°C, 40°C, 45°C, and 50°C, and the remaining factors were set as: substrate mass fraction 2%, enzymatic hydrolysis time 1 h, enzyme dosage 6000 U / g, and pH 3. The peptide yields and effects on NO secretion by RAW264.7 cells of the enzymatic hydrolysis solutions at different enzymatic hydrolysis temperatures were compared to determine the optimal enzymatic hydrolysis temperature.

[0031] (4) Influence of solid-liquid ratio The collagen:pure water ratio was set at 1:20, 1:30, 1:40, 1:50, and 1:100, and the remaining factors were set as follows: enzymatic hydrolysis time 1 h, enzymatic hydrolysis temperature 37°C, enzyme dosage 6000 U / g, and pH 3. The peptide yields and effects of enzymatic hydrolysis solutions with different substrate mass fractions on NO secretion by RAW264.7 cells were compared to determine the optimal substrate mass fraction for enzymatic hydrolysis.

[0032] 1.7 Response surface optimization experimental design Through the determination of the above single-factor experiment, the three factors of enzyme dosage, material-liquid ratio and temperature were selected as independent variables, the NO concentration secreted by RAW264.7 cells was used as the response value, and a three-factor three-level experiment was selected. The experimental design was carried out according to the Box-Behnken (BBD) principle. Design-Expert 13 was used to conduct a three-factor three-level experiment to determine the optimal conditions for fish scale enzymatic hydrolysis. The experimental design is shown in the following table.

[0033] Table 1. Factor levels in the Box-Behnken experiment 1.8 Ultrafiltration of enzymatic hydrolysate The ultrafilter was cleaned with 0.1 mol / L sodium hydroxide solution to separate the components <3 kDa, which were then freeze-dried for storage.

[0034] 1.9 RAW264.7 cell proliferation assay A cell suspension was seeded at a density of 3x105 cells / mL in a 96-well plate, with 100 μL / well added for 24 hours. The supernatant was discarded, and samples ultrafiltered from the 1.8-well enzymatic hydrolyzate were added at varying concentrations (0, 3.125, 6.25, 12.5, 25, 50, 100, 200, and 400 μg / mL). Peptides (PDPFRMY, GPQGPIGPR, and GPSGPP) were added at concentrations of 50, 100, 200, 400, and 800 μg / mL, respectively). 100 μL was added to each well. For the LPS model group, 1 μg / mL LPS was added, and the cells were incubated for 24 hours. The supernatant was discarded, and 100 μL of 10% CCK-8 solution was added. Protect from light. Incubate for 1 hour, and absorbance was measured at 450 nm using a microplate reader.

[0035] Cell survival rate (%) = (absorbance A of the experimental group - absorbance A of the zero-adjustment group) / (absorbance A of the control group - absorbance A of the zero-adjustment group) * 100 1.10 Neutral red uptake assay in RAW264.7 cells RAW 264.7 cells (3 x 105 cells / ml) were cultured in a 96-well plate for 24 hours. The supernatant was discarded and the cells were incubated with various concentrations of sample (0, 50, 100, 200, and 400 μg / mL), LPS (1 μg / mL), and complete culture medium for 24 hours. The culture medium was then aspirated and 100 μL of 0.1% neutral red solution (prepared in saline: 0.1 g neutral red in 100 mL of saline) was added, followed by an additional 0.5 hour incubation. The supernatant was discarded, and the cells were washed twice with PBS and then incubated in 100 μL of a cell lysis buffer consisting of acetic acid and anhydrous ethanol (v / v = 1:1) for 2 hours. Absorbance was measured at 540 nm using a microplate reader.

[0036] Phagocytosis rate (%) = OD value of experimental group / OD value of control group * 100 1.11 Determination of NO secretion by RAW264.7 cells Treat cells as in the neutral red uptake experiment. Add 50 μL of cell supernatant to a 96-well plate. Add 50 μL of room temperature Griess Reagents 1 and 2 to each well in sequence. Measure absorbance at 540 nm and use the curve to determine the NO concentration.

[0037] 1.12 Observing cell status A cell suspension was seeded in a 6-well plate at a density of 1x106 cells / mL, 2 mL / well, and incubated for 24 hours. The supernatant was discarded, and 2 mL of sample at different concentrations (0, 50, 100, and 400 μg / mL) was added to each well. For the LPS model group, 1 μg / mL of LPS was added, and the cells were incubated for 24 hours. Cell morphology was observed and photographed using an inverted microscope.

[0038] 1.13 Determination of Amino Acid Composition of Collagen Peptides Take 0.1g of the sample and add 10ml of 6M hydrochloric acid. Fill the container with nitrogen and seal. Digest at 105°C for 22h. After cooling, add water to 50ml. Take 1ml of the above solution and blow dry with nitrogen. Reconstitute with 1ml of water and blow dry. Reconstitute with 1ml of 0.02M hydrochloric acid, filter through a 0.22μm filter, and filter into a Hitachi LA8080 instrument with an injection volume of 20µL. 1.14 Collagen peptide Fourier infrared spectroscopy FTIR spectra of the lyophilized product (powder) of tilapia scale <3 kDa collagen peptide were recorded using a Nicolet IZ10 FTIR instrument from Thermo Fisher Scientific (Waltham, MA, USA). The spectral resolution was 4 cm-1, and 32 scans were acquired in the range of 350-7800 cm-1.

[0039] 1.15 Peptide Sequencing by LC-MS / MS Tilapia scale gelatin hydrolysate was dissolved in 0.1% formic acid and analyzed by LC-MS / MS using an EASY-nLC 1000 ultra-high-performance nano-LC system (Thermo Scientific, USA) coupled with a QE high-resolution mass spectrometer (Thermo Scientific, USA). A PepMap RSLC C18 analytical column (75 μm × 150 mm, 2 μm, 100Å, Thermo Scientific, USA) was used. The mobile phase consisted of ultrapure water containing 0.1% formic acid (phase A) and acetonitrile containing 0.1% trifluoroacetic acid (phase B). The gradient elution program was as follows: 5-10% phase B (0-8 min), 10-40% phase B (8-50 min), 40-95% phase B (50-60 min), 95% phase B (60-65 min), and 95-5% phase B (65-70 min). The flow rate was 0.25 μL / min, and the injection volume was 3 μL. The mass spectrometer scan range was 300–1800 m / z, with a resolution of 70,000. HCD fragmentation was performed on the top 20 most intense peaks (normalized collision energy 27%). LC-MS / MS data were searched against the uniprot-taxonomy_id8139 database using PEAKS software. The parameters were set as follows: nonspecific enzyme cleavage, oxidation and acetylation as variable modifications, precursor ion mass tolerance of 10 ppm, and secondary fragment ion mass tolerance of 0.02 Da.

[0040] 1.16 Screening of immunopotent peptides This study conducted a preliminary screening of 1,171 peptides from tilapia scale collagen peptides <3 kDa, as determined by LC-MS / MS. The criteria included amino acid number ≤ 10, -10LgP ≥ 40, and PeptideRanker > 0.8. Excessive hydrophobicity prevented molecular dissolution and poor absorption. Excessive water solubility prevented absorption by penetrating the hydrophobic regions of cell membranes, thus requiring appropriate hydrophilicity. Peptide solubility is related to its isoelectric point, so its isoelectric point was predicted (http: / / pepcalc.com / ). The peptides were further screened: checking whether the peptides were reported in the BIOPEP database (https: / / biochemia.uwm.edu.pl / biopep-uwm / ), predicting whether the peptides had potential toxicity (https: / / webs.iiitd.edu.in / raghava / toxinpred), whether they had allergenicity (https: / / webs.iiitd.edu.in / raghava / algpred2 / batch_action.php), and using AutoDock Vina to simulate the binding energy of the peptides to TLR4 / MD-2.

[0041] 1.17 Statistics All experiments were repeated three times, and data are presented as mean ± standard deviation. GraphPad Prism 10 software was used for data analysis and graphing. One-way analysis of variance (ANOVA) was performed on the experimental data, and P < 0.05 was considered statistically significant.

[0042] Result Analysis (1) Collagen extraction Based on the weight of delimed fish scales, the collagen extraction rate is .

[0043] (II) Screening of proteases Table 2 Enzyme activity, hydrolysis conditions and addition amount of various proteases Table 1 shows the enzymatic hydrolysis conditions and addition amounts of papain, alkaline protease, trypsin, pepsin, and neutral protease. The peptide yield is used as an indicator to monitor the degree of collagen hydrolysis. The results are as follows: Figure 1 As shown; the production of active peptides was evaluated by enzymatic hydrolysis of RAW264.7 cells for NO secretion, and the results were as follows: Figure 2 shown.

[0044] The peptide yields of trypsin and neutral protease varied most significantly during the first 60 minutes, remaining stable from 60 to 270 minutes. The yield of trypsin peaked at 40.1632% at 180 minutes, while the yield of neutral protease peaked at 22.743% at 270 minutes. Alkaline protease and papain showed a significant upward trend during the first 40 minutes, reaching their maximum at 40 minutes, before declining slightly and then stabilizing. The yields of alkaline protease and papain peaked at 27.9178% and 33.8807% respectively at 40 minutes. The peptide yield of pepsin showed significant variation during the first 20 minutes, as pepsin has an optimal pH of 3.5-4.5, and the acidic environment rapidly dissolves collagen. Peptide yields from pepsin did not change significantly after 20 minutes, peaking at 42.3847% at 270 minutes.

[0045] The peptide yields from collagen hydrolysis by the five proteases were ranked in descending order: pepsin > trypsin > papain > alkaline protease > neutral protease. The effects of different enzyme hydrolysis solutions on NO secretion by RAW264.7 cells revealed no difference in NO secretion compared to the blank control when treated with the five hydrolysis solutions at a 100-fold dilution. However, NO secretion by RAW264.7 cells treated with the 10-fold dilution was significantly higher than that of the blank control (p < 0.05). The 10-fold dilution of pepsin significantly increased NO secretion by RAW264.7 cells (14.2674 μM) compared to the other protease and LPS treatment groups (11.9236 μM) (p < 0.0001). Therefore, pepsin was selected for the next experiment.

[0046] (III) Single-factor experimental results like Figure 3 As shown, the NO concentration stimulated by the enzymatic hydrolysis solution gradually decreased over time in RAW264.7 cells. The NO concentration did not change significantly within the first hour and was significantly higher than that at 2 and 3 hours (P < 0.05). The NO concentration peaked at 18.3762 μmol / L at 20 minutes. During the first hour of the reaction, the protein was hydrolyzed into active peptides. Over time, the active peptides were hydrolyzed into inactive peptides, and the NO concentration decreased. With time, the peptide concentration initially increased and then stabilized. Therefore, the optimal enzymatic hydrolysis time is 20 minutes.

[0047] As the enzyme dosage increased from 1000 U / g to 12000 U / g, the peptide yield initially increased and then decreased, reaching a peak of 41.9856% at 3000 U / g. The increase in enzyme dosage accelerated protein hydrolysis. Further increases in enzyme dosage resulted in protein hydrolysis to amino acids, and the peptide yield decreased. With increasing enzyme dosage, the NO concentration initially increased and then gradually decreased, reaching a peak of 12.7396% at 3000 U / g. Therefore, 3000 U / g was selected as the optimal enzyme dosage, and levels of 1000 U / g, 3000 U / g, and 5000 U / g were selected for response surface optimization experiments.

[0048] Within the temperature range of 30-50°C, peptide yield gradually increased and then stabilized, reaching a peak of 40.7346% at 40°C, significantly higher than the 37.9631% yield at 35°C (P < 0.05). Increasing temperature facilitates collagen dissolution and accelerates the enzymatic hydrolysis reaction, thereby increasing peptide yield. The 40°C enzymatic hydrolysis solution caused RAW264.7 cells to secrete nitric oxide (NO) at a concentration of 15.4479 μmol / L, significantly higher than the 13.9549 μmol / L NO concentration at 35°C (P < 0.05). Therefore, 40°C was selected as the optimal temperature, and levels at 35°C, 40°C, and 45°C were selected for response surface optimization experiments.

[0049] As the ratio of pure water to solvent increased from 1:20 to 1:100, the peptide yield gradually increased, reaching a maximum of 45.1986% at a solid-to-liquid ratio of 1:100 (p < 0.05). This may be because the increase in solvent facilitates contact and mobility between the enzyme and substrate, decreasing solution viscosity, accelerating the hydrolysis reaction and producing more active peptides. The NO concentration reached a maximum of 17.6007 μmol / L at 1:30, significantly higher than the 12.2188 μmol / L at 1:20 (p < 0.05), and did not differ significantly from the NO concentrations at 1:40, 1:50, and 1:100 (p > 0.05). Therefore, 1:30 was selected as the optimal solid-to-liquid ratio, and levels of 1:20, 1:30, and 1:40 were selected for response surface optimization experiments.

[0050] (IV) Response surface optimization results (1) Response surface design and regression model variance analysis Table 3 Response surface experimental design and results Table 4 Analysis of variance of regression model Design-Expert 13 software was used to analyze the data in Table 1. The resulting quadratic regression equation for the effect of enzymatic hydrolysate on NO secretion in RAW264.7 cells was Y = 16.15 + 0.1584A + 0.1584B - 0.0347C - 0.1215AB - 0.1519AC + 0.3168BC - 0.7005A² - 0.8134B² - 0.0278C². The analysis of variance for the regression model is shown in Table 2. The model p = 0.0011 < 0.01, indicating a highly significant model. The lack-of-fit p = 0.6923 > 0.05 indicates a small error between the actual value and the error value, indicating a good fit. The interaction between BC and A2 on NO concentration was significant (p < 0.05), and A2 and B2 had significant effects on NO concentration (p < 0.05). The other factors had no significant effects on NO concentration. The lack of significant effects of individual factors suggests that the effects of each factor on NO concentration are not linear. The F value is an important indicator of the impact of each factor on the response value. A larger F value indicates a greater influence on the response value. The F values of A and B are both greater than those of C. Therefore, the effects of enzyme dosage and material-to-liquid ratio on NO concentration are greater than those of temperature. R² = 0.9476, R² Adj = 0.8802. The closer the R² is to 1, the better the model fits the data.

[0051] (2) Response surface plot analysis The contour lines are elliptical, indicating that the interaction has a significant effect on NO concentration, and the contour lines are circular, indicating that the interaction is not significant. Figure 4-Figure 6 The contour plots for the interactions between A and C, and B and C, exhibit elliptical shapes. The interaction between B and C significantly impacted NO concentration (p<0.05). The steepness of the slope corresponding to each factor in the 3D response surface plot reflects the degree of influence of that factor on the response. The interaction plots for BC and AC reveal that the slopes for the solid-liquid ratio and enzyme dosage are steeper than those for temperature. Therefore, the solid-liquid ratio and enzyme dosage have a greater impact on the response than temperature, which is consistent with the results of the model analysis of variance.

[0052] (V) Optimal conditions and verification The optimal process parameters were obtained by optimizing the software Design-Expert 13: material-liquid ratio 1:28.3575 g / mL, enzyme dosage 4802.39 U / g, and temperature 35°C. At this time, the NO concentration was the highest, and its theoretical value was 15.8873 μmol / L. Under these conditions and in combination with the actual selection: material-liquid ratio 1:28 g / mL, enzyme dosage 4800 U / g, and temperature 35°C. Experimental verification was carried out and the experiment was repeated three times. The experimental results showed that the NO concentration was , which has no significant difference from the predicted value of the regression equation, indicating that the model is feasible.

[0053] (VI) Effect of collagen peptide on RAW264.7 cell proliferation CCK-8 assays are commonly used to detect cell viability. Within the range of 3.125 μg / mL to 400 μg / mL, cell viability gradually increased. The lowest concentration of 25 μg / mL showed a significant difference in cell viability (139.19%) compared to the control group (p<0.05). The highest cell viability reached 182.91% at 400 μg / mL. Figure 7 Therefore, collagen peptides <3 kDa are non-toxic and can be used for in vitro immune enhancement validation.

[0054] (VII) Effect of collagen peptide on the phagocytic ability of RAW264.7 cells The enhanced phagocytic ability indicates that macrophages are activated. Figure 8 As shown in the results, treatment with collagen peptides <3 kDa at concentrations of 50-400 μg / mL can enhance the phagocytic ability of macrophages, with the highest phagocytic ability reaching 123.61% at 200 μg / mL. Therefore, treatment with collagen peptides <3 kDa can enhance the phagocytic ability of cells.

[0055] (8) Effect of collagen peptide on NO secretion in RAW264.7 cells like Figure 9As shown in the results, NO concentration increased with the increase of collagen peptide concentration. At the lowest concentration of 100 μg / mL, the NO concentration (2.03 μM) was significantly different from the control group (p<0.05). At 400 μg / mL, the NO concentration reached a maximum of 9.60 μM. Therefore, collagen peptides <3 kDa have immune-enhancing activity at low concentrations.

[0056] (IX) Effects of collagen peptides on RAW264.7 cell morphology In the control group, RAW264.7 cells were round or oval and had no tentacles. After LPS treatment, RAW264.7 cells differentiated and had irregular morphology with pseudopodia and tentacles. The density of RAW264.7 cells treated with <3kDa collagen peptide increased significantly compared with the control group, which was consistent with the CCK-8 results. The cells were round, with few irregularly shaped cells, e.g. Figure 10 shown.

[0057] (10) Analysis of Amino Acid Composition of Collagen Peptides As shown in Table 5, the essential amino acid content of tilapia scale <3kDa collagen peptides is 7.69g / 100g, accounting for 24.98% of the total amino acids. The amino acids with higher concentrations are Gly, Glu, and Pro, accounting for 46.76% of the total amino acid content. This is consistent with the "Gly-XY" structure of collagen, where X and Y are typically proline and hydroxyproline. Studies have shown that hydrophobic amino acids are characteristic of immunomodulatory peptides. The hydrophobic amino acid content is 17.58g / 100g, accounting for 57.12% of the total amino acids. The branched-chain amino acids Leu, Val, and Ile are present at 1.45, 1.19, and 0.43g / 100g, respectively, accounting for 4.71, 3.87, and 1.40% of the total amino acids, respectively. Therefore, tilapia scale <3kDa collagen peptides have the potential to enhance immune activity.

[0058] Table 5 Amino acid sequence of tilapia scale <3 kDa collagen peptide (11) Collagen peptide Fourier infrared spectroscopy analysis like Figure 11 As shown in Figure 2, the FTIR spectrum of tilapia scale collagen peptide has characteristic absorption peaks of amide A, B, I, II, and III. Amide A is usually caused by NH stretching vibration. The broad absorption peak of amide A of tilapia scale collagen peptide is 3369.75 cm -1 The broadening of this band is related to the increase of peptide chains, indicating that collagen is more degraded. −1 The absorption peak of tilapia scale collagen peptide amide B is 2960.25 cm -1Amide I occurs at a wave number of 1600–1700 cm -1 , which is mainly related to the NH stretching and hydrogen bonding between C=O. The absorption peak of tilapia scale collagen peptide amide I is 1650.08 cm -1 The study showed that the amplitude of the amide I peak decreased, then became broader, and an additional shoulder appeared, which was caused by the collagen being heated at a higher temperature. Amide II generally appears at 1550-1600 cm -1 The absorption peak of tilapia scale collagen peptide amide II is 1407.95 cm -1 Amide III appears at 1200 ~ 1400 cm -1 The absorption peak of tilapia scale collagen peptide amide III is 1232.62 cm -1 The amide III peak height is 1450 cm -1 The absorbance ratio of peak heights is often used to determine the triple helical structure of collagen. For tilapia scale collagen peptide, this ratio is 0.92, close to 1, indicating the presence of a triple helical structure.

[0059] (XII) Analysis of collagen peptide LC-MS / MS sequencing results Tilapia scale collagen was hydrolyzed with pepsin to obtain active peptides. The hydrolyzate was ultrafiltered using a 3 kDa membrane, and the <3 kDa fraction was subjected to LC-MS / MS for peptide sequence identification (conditions similar to those in 1.15). A total of 1,171 peptides were identified from the <3 kDa fraction of the tilapia scale collagen hydrolyzate. Among the identified peptides, 4 (0.34%), 57 (4.87%), 445 (38%), and 665 (56.79%) peptides had molecular weights >3,000 Da, 2,000-3,000 Da, 1,000-2,000 Da, and <1,000 Da, respectively. The molecular weights of the identified peptides were primarily less than 2,000 Da. There are 38 peptides containing 3 amino acids, 98 peptides containing 4 amino acids, 187 peptides containing 5 amino acids, 114 peptides containing 6 amino acids, 77 peptides containing 7 amino acids, 72 peptides containing 8 amino acids, 94 peptides containing 9 amino acids, and 491 peptides containing 10 amino acids, such as Figure 12 shown.

[0060] (13) Analysis of screening results of immunopotentiating peptides Based on the number of amino acids <10, 738 peptides were identified from the 1171 peptides. Based on the -10LgP ≥40, 219 peptides were identified from the 738 peptides. Based on the peptide ranker >0.8, 18 peptides were identified from the 219 peptides. The water solubility, isoelectric point, and hydrophobicity of these 18 peptides were predicted. Only PGR has been reported to exhibit dipeptidyl peptidase IV inhibitory (DPP IV) and ACE inhibitory activity; the remaining peptides were identified from initial screening. All 18 peptides were predicted to be non-toxic and non-allergenic. AutoDock Vina was used to simulate the binding of each of the 18 peptides to TLR4 / MD-2. Affinity represents the change in the free energy of ligand-receptor binding, expressed in kcal / mol. Negative values indicate spontaneous binding, and smaller values indicate stronger binding. Affinity typically ranges from -5 to -15 kcal / mol. -5 to -7 kcal / mol indicates moderate binding strength. -7 to -10 kcal / mol: Strong binding strength. -10 to -15 kcal / mol: Very strong binding strength. Twelve peptides were found to have strong binding, and six to have moderate binding. Based on binding energies greater than or equal to -8 kcal / mol, four peptides were identified: PDPFRMY, GPQGPIGPR, PFRMY, and GPSGPP, with binding energies of -8.6, -8.4, -8, and -8 kcal / mol, respectively. Because PDPFRMY and PFRMY contain repetitive sequences, these three peptides were selected for chemical synthesis to further investigate their immunopotency.

[0061] Table 6 Amino acid sequences of tilapia scale collagen peptides with PeptideRanker > 0.8 and molecular weight < 3 kDa Depend on Figure 13 As shown, PDPFRMY at a concentration of 800 μg / mL promoted the proliferation of RAW264.7 cells by a maximum of 150.373%, which was significantly different from the control group (p<0.01). There was no significant difference between GPQGPIGPR and GPSGPP at concentrations of 50-800 μg / mL compared with the control group, indicating that PDPFRMY was the best at enhancing immune activity.

Claims

1. A tilapia scale collagen peptide, characterized in that: The tilapia scale collagen peptide is composed of the following peptide segments: PDPFRMY, GPQGPIGPR, PFRMY, GPSGPP, APDPFR, KAPDPFRMY, PGPGPMGL, DPGPGPMG, DPFRMY, PGGLP, PGPGMP, GPFS, FGAG, PLGP, GPLG, PGR, GPAGGL, PGGGF.

2. A method for preparing tilapia scale collagen peptide according to claim 1, characterized in that: The following steps are involved: (1) Soaking tilapia scales in a citric acid solution, ultrasonically treating the scales, cleaning the scales until neutral, filtering, and drying to obtain de-ashed scales; (2) Mixing dried de-limed fish scales with deionized water, heating the mixture, filtering and concentrating it to obtain gelatin; (3) Add gelatin to pure water, mix well, add enzyme, and perform enzymatic hydrolysis. After the enzymatic hydrolysis is completed, inactivate the enzyme, cool to room temperature, and centrifuge to collect the supernatant to screen tilapia scale collagen peptides.

3. The preparation method according to claim 2, characterized in that In step (1), the ratio of the tilapia scales to the 4% citric acid solution is 1:10 (W / V); and the ultrasonic treatment is performed at a power of 40 W for 90 min.

4. The preparation method according to claim 2 or 3, characterized in that In step (2), the ratio of the delimed fish scales to deionized water is 1:15 (W:V); the pH is adjusted to 5.5; and the heating is performed at 80° C. for 2 h.

5. The preparation method according to claim 2 or 4, characterized in that In step (3), the material-liquid ratio of gelatin to water is 1:20-100; and the amount of enzyme added is 1000-12000 U / g.

6. The preparation method according to claim 5, characterized in that In step (3), the enzyme is alkaline protease, papain, trypsin, pepsin or neutral protease.

7. The preparation method according to claim 2, 5 or 6, characterized in that: In step (3), the enzymatic hydrolysis is carried out at 35-45°C and pH 3-10 for 4.5 hours.

8. The preparation method according to any one of claims 2 to 7, characterized in that Tilapia scale collagen peptides were screened from enzymatic hydrolysates with PeptideRanker >0.8 and molecular weight <3kDa.

9. Use of the tilapia scale collagen peptide according to claim 1 in preparing a product that enhances immune activity.

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