Sliced duck antioxidant peptide as well as preparation method and application thereof

Abstract

The invention relates to pressed salted duck antioxidant peptide and a preparation method thereof. The method comprises the following steps: carrying out synergistic inoculation and fermentation treatment on lactobacillus curvatus and staphylococcus mimicus, and carrying out pickling, air drying and peptide extraction processes to obtain the small molecular peptide with the molecular weight of less than 10 kDa. Through nano-LC-MS / MS (Liquid Chromatography-Mass Spectrometry / Mass Spectrometry) detection, computer virtual screening and molecular docking, three peptide sequences, namely GIRLGLDPKL, PAPAAKAGASTGRIV and PEKPPTIDWA, with remarkable antioxidant activity are determined. According to the method, protein degradation can be effectively promoted, the yield of the antioxidant peptide is increased, and the obtained peptide has good stability and safety and is suitable for development of functional food and health care products.

Description

Technical Field

[0001] This invention relates to the field of antioxidant peptide technology, specifically to a salted duck antioxidant peptide, its preparation method, and its application. Background Technology

[0002] Chinese salted duck, a traditional fermented meat product, boasts a production history spanning hundreds of years due to its unique curing and air-drying processes and distinctive regional flavor. In recent years, with the popularization of functional foods and nutritional health concepts, research on fermented meat products has gradually expanded from flavor and safety to exploring nutritional value and health functions. In salted duck, muscle protein can be gradually degraded into peptides and free amino acids under the action of endogenous enzymes and exogenous proteases from microorganisms. These low-molecular-weight compounds not only participate in flavor formation but may also possess certain biological activities. Antioxidant peptides, in particular, have received widespread attention in recent years due to their ability to scavenge free radicals, inhibit lipid peroxidation, and delay food oxidative deterioration. Compared with chemically synthesized antioxidants, antioxidant peptides derived from natural food proteins are safer and possess physiological functional activity, making them a research hotspot in food science, functional foods, and nutritional products. Existing studies have shown that during fermentation processes involving dominant strains such as lactic acid bacteria and coagulase-negative staphylococci, the degree of protein hydrolysis and peptide production are significantly increased, releasing more peptide sequences with potential antioxidant activity. Therefore, exploring the unique antioxidant peptide resources in salted duck not only helps to enhance the nutritional value of the product, but also provides a new research direction for exploring the modern applications of traditional meat products.

[0003] However, existing technologies for utilizing duck protein derivatives as antioxidant functional components still have several shortcomings: First, most current research focuses on the quality control and flavor improvement of duck, with insufficient systematic research on protein degradation mechanisms and the formation patterns of antioxidant peptides, resulting in unclear types and structural characteristics of antioxidant active peptides. Second, the generation of peptides under natural fermentation conditions is random and unstable, restricting the targeted acquisition and stable application of active ingredients. Third, although some studies have found increased peptide content and antioxidant capacity through inoculation with specific fermentation strains, there is still a lack of screening and identification of key functional peptide sequences and elucidation of their mechanisms of action. Existing processes often remain at the level of crude peptide functional assessment, failing to achieve the isolation, preparation, and activity mechanism verification of specific antioxidant peptides. This prevents the full development and utilization of the potential antioxidant resources in duck, limiting their application in functional foods, nutritional supplements, and even the development of natural antioxidants. Therefore, there is an urgent need for a technical solution that can effectively prepare and apply antioxidant peptides from duck protein to achieve in-depth exploration and high-value-added utilization of the functional value of traditional meat products. Summary of the Invention

[0004] To achieve the above and related objectives, the present invention provides a duck antioxidant peptide, the amino acid sequence of which is at least one of GIRLGLDPKL, PAPAAKAGASTGRIV, and PEKPPTIDWA, as shown in SEQ ID NO: 1-3.

[0005] In addition, this application also provides a method for preparing antioxidant peptides from preserved duck, comprising the following steps: The first step is raw material marinating: Select slaughtered and bled ducks, remove feathers and internal organs, and evenly coat the surface and abdominal cavity with 6% salt by weight. Let it stand at 4℃ for 24 hours to allow the salt to fully penetrate. Then, place the marinated duck carcass in a saturated salt solution for another 12 hours to further enhance the marinating effect. After marinating, remove the duck carcass and place it in a well-ventilated environment to allow the surface moisture to drain naturally, thus obtaining the pre-marinated duck raw material.

[0006] The second step is the preparation of the bacterial suspension: *Lactobacillus curvaturei* and *Staphylococcus mimicus*, isolated and identified from traditional salted duck, were inoculated into MRS liquid medium and NB liquid medium, respectively. After incubation at 37°C for 24 hours, they were subcultured for another 12 hours to obtain viable bacterial cultures. After incubation, the fermentation broth was centrifuged at 8000 rpm for 10 minutes to collect the bacterial cells. The cells were washed three times with 0.85% sterile physiological saline and finally resuspended in physiological saline to achieve a final concentration of 1×10⁻⁶. 7 The concentration of cfu / mL was collected and stored at 4°C for later use.

[0007] The third step is bacterial inoculation: The two bacterial suspensions prepared above are mixed at a volume ratio of 1:1 and evenly inoculated onto the surface and inside the muscle tissue of the cured duck using a combination of spraying and injection methods to ensure that the bacterial strains are evenly distributed inside and outside the duck. This inoculation method can promote the rapid colonization of lactic acid bacteria and staphylococci and ensure the full progress of protein hydrolysis reactions during subsequent fermentation.

[0008] The fourth step is fermentation and air drying: After inoculation, the duck carcasses are first placed in an environment of 25℃ and 65% relative humidity to air dry for one day to promote surface moisture evaporation and initial bacterial colony growth; then, the duck carcasses are moved to a temperature of 18-20℃ and relative humidity of 65% to continue air drying for 9 days, allowing it to gradually complete the fermentation and maturation process. After this process, a fermented pressed duck product with typical flavor and texture characteristics can be obtained.

[0009] Step 5, Crude Peptide Extraction: Take fermented, mature duck muscle tissue and add 0.01M hydrochloric acid solution at a mass ratio of 1:4. Homogenize at 12000 rpm for 3 minutes under ice bath conditions using a high-speed homogenizer, then let it stand at 4℃ for 2 hours. Filter the resulting mixture through double-layer gauze to remove solid residue. Centrifuge the filtrate at 12000 rpm for 20 minutes and collect the supernatant. Fractionate the supernatant through an ultrafiltration membrane with a molecular weight cutoff of 10 kDa, collecting the small molecule peptide fractions with a molecular weight less than 10 kDa. Dry these fractions in a vacuum freeze dryer to obtain crude duck peptide powder.

[0010] Step 6: Obtaining the target antioxidant peptides: The crude peptide powder from preserved duck was analyzed using nano-LC-MS / MS, and the resulting peptide sequences were functionally predicted and their activity evaluated using bioinformatics methods. Active peptides were screened using PeptideRanker and the BIOPEP-UWM database, and their binding affinity to Keap1 protein was verified using molecular docking technology. Ultimately, three novel preserved duck antioxidant peptides were identified: GIRLGLDPKL, PAPAAKAGASTGRIV, and PEKPPTIDWA.

[0011] The antioxidant peptides from preserved duck of the present invention can be used in food, especially in the preparation of food preservatives.

[0012] The antioxidant peptides from preserved duck of the present invention can be used in food, especially in the preparation of functional foods or nutritional supplements.

[0013] The antioxidant peptides from preserved duck of the present invention can be used in food, especially in the preparation of antioxidants.

[0014] The beneficial effects of this application are that co-inoculation with *Lactobacillus curvularis* and *Staphylococcus mimicus* reduces the pH, water activity, and moisture content of cured duck. Furthermore, the inoculation fermentation significantly increases the number of microorganisms and protease activity during the fermentation process, thereby significantly improving the protein hydrolysis index of the cured duck. During the fermentation process, the content of peptides, DPPH free radical scavenging rate, ABTS free radical scavenging rate, and Fe2+ after inoculation fermentation are also significantly improved. 2+The chelation capacity was significantly higher than that of natural fermentation. Furthermore, peptidomics was used to analyze the changes in peptide profiles at the end of fermentation under inoculated fermentation and natural fermentation. The results showed that the number of peptides produced under inoculated fermentation was far greater than that under natural fermentation, with a higher proportion of peptides smaller than 3000 Da. This reveals that inoculated fermentation can promote the formation of small molecule peptides in preserved duck. In addition, this study analyzed GO annotation and KEGG metabolic pathway analysis of differentially expressed peptide-derived parental proteins. Functional prediction analysis of peptides revealed that inoculated fermentation increased the proportion of potentially bioactive peptides. Among them, the number of peptides with antioxidant activity also increased significantly. Furthermore, three novel antioxidant peptides (GIRLGLDPKL, PAPAAKAGASTGRIV, and PEKPPTIDWA) were screened through computer-generated virtual screening and molecular docking. Molecular docking further analyzed the molecular mechanism by which these three peptides may exert antioxidant activity in vivo. In conclusion, this study provides important insights into the effects of *Lactobacillus curvaturei* and *Staphylococcus mimicus* on the degradation of preserved duck proteins and the preliminary formation of antioxidant peptides, and also provides a theoretical basis for developing preserved duck products with nutritional value and health benefits. Attached Figure Description

[0015] Figure 1 (A) Changes in protease activity; (B) Changes in the protein hydrolysis index; Figure 2 (A) Changes in peptide content; (B) Changes in DPPH free radical scavenging rate; (C) Changes in ABTS free radical scavenging rate; (D) Fe 2+ Changes in chelating ability; Figure 3 Mass spectrometry analysis; (A) Venn diagram of identified peptides; (B) Venn diagram of parental proteins; (C) peptide chain length distribution; (D) molecular weight distribution; (E) N-terminal amino acid percentage; (F) C-terminal amino acid percentage; Figure 4 Identifying the distribution of parental proteins of peptides; (A) Control group; (B) Inoculated group; (C) Hierarchical clustering analysis heatmap of parental proteins (top 20 proteins from which peptides originate); Figure 5 (A) GO enrichment analysis; (B) KEGG pathway analysis; Figure 6 Distribution of bioactive peptides; (A) Venn diagram of bioactive peptides; (B) Distribution of functional peptides in the control group; (C) Distribution of functional peptides in the inoculated group; Figure 7 Molecular docking results of peptides with Keap1; (A) GIRLGLDPKL; (B) PAPAAKAGASTGRIV; (C) PEKPPTIDWA. Detailed Implementation

[0016] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in these embodiments.

[0017] I. Preparation of Salted Duck Preparation of the starter culture: Lactobacillus curvatus D2 and Staphylococcus simulans D12 were derived from traditional preserved duck. The Lactobacillus curvatus D2 has been deposited at the China Center for Type Culture Collection (CCTCC) on February 21, 2023, with the accession number CCTC CN O: M2023162. The Staphylococcus simulans D12 has also been deposited at the China Center for Type Culture Collection (CCTCC) on February 21, 2023, with the accession number CCTCC NO: M2023163. L. curvatus D2 and S. simulans D12 were inoculated into Man Rogasa Sharpe (MRS) broth and nutritious broth (NB), respectively, and cultured at 37 °C for 24 h, followed by subculturing at 37 °C for 12 h. Bacteria were collected by centrifugation (8000 rpm, 10 min), washed three times with 0.85% sterile physiological saline, and finally resuspended in sterile physiological saline to obtain a final concentration of 10. 7 The bacterial suspension of cfu / mL was stored at 4 ℃ for later use.

[0018] Preparation of the preserved duck: After slaughtering, marinate the duck in saturated brine at 4℃ for 24 hours, then place it in a well-ventilated room to drain excess surface moisture. Next, inoculate the marinated duck with a bacterial solution at a rate of 10... 7 cfu / g meat. This study set up two experimental groups: (1) uninoculated ducks (control group); (2) ducks inoculated with L. curvatus D2 and S. simulans D12 (inoculation group) at a volume ratio of 1:1. Subsequently, the ducks were air-dried at 25℃ and 65% relative humidity for 1 day, and then air-dried at 18-20℃ and 65% relative humidity for 9 days to obtain the final duck product. Samples were taken on days 0, 1, 4, 7 and 10 during the air-drying process.

[0019] II. Testing Methods 2.1 Microbial Analysis In a sterile environment, 10g of preserved duck sample was weighed and placed in a sterile homogenization bag containing 90mL of sterile physiological saline. The sample was homogenized for 3 minutes using a sterile homogenizer. Then, 10-fold dilutions were prepared continuously, and 0.1ml of each dilution was inoculated onto specific culture media for the following analyses: lactic acid bacteria were incubated on MRS medium at 30°C for 48h; staphylococci were incubated on MSA medium at 30°C for 48h; and total bacterial count was incubated on PCA medium at 30°C for 48h.

[0020] 2.2 Determination of physicochemical properties The pH value of the salted duck was determined using a pH meter. The water activity of the mixture was measured using a portable water activity meter.

[0021] 2.3 Protein hydrolysis index (PI) determination The total protein content in the salted duck extract was determined using the BCA method. Simultaneously, the fluorescence intensity of the N-terminal α-amino groups of the fluorescently labeled peptides and amino acids was measured using a fluorophotometer under excitation wavelengths of 375 nm and emission wavelengths of 475 nm. PI (%) was defined as the percentage of N-terminal α-amino groups in the total protein content of the salted duck sample.

[0022] 2.4 Protease activity assay Accurately weigh 3g of chopped duck meat and mix it with 30mL of phosphate buffer. Homogenize the mixture at 12000rpm and 4℃ for 3min. Then, centrifuge the homogenate (1200rpm, 10min) to collect the supernatant and store it at 4℃ for the next step of enzyme activity assay.

[0023] 1 mL of crude enzyme solution was mixed with 1 mL of 2% casein solution preheated at 40 °C and incubated at 40 °C for 10 min. Then, 2 mL of trichloroacetic acid (0.4 M, TCA) solution was added to the mixture to terminate the reaction, and the supernatant was collected by centrifugation (12000 rpm, 10 min). 1 mL of the supernatant was then mixed with 5 mL of Na₂CO₃ (0.4 M) and 1 mL of Folin-Ciocalteu solution and incubated at 40 °C for 20 min. The control group samples were treated using the same procedure, but trichloroacetic acid solution was added before mixing the crude enzyme solution with the casein solution. Finally, the absorbance of the samples was measured at 680 nm using a microplate reader. One unit of enzyme activity (expressed as U / g) is defined as the production of 1 μg of tyrosine per minute of casein hydrolysis at 40 °C.

[0024] 2.5 Peptide extraction and peptide content determination Add 200 mL of 0.01 M HCl to 50 g of cured duck meat and homogenize at 12000 rpm for 3 min. Let the homogenate stand at 4℃ for 2 h. Then, filter the homogenate through two layers of gauze and centrifuge at 12000 rpm for 20 min. Obtain peptides with a molecular weight less than 10 kDa by passing the supernatant through an ultrafiltration centrifuge tube. Freeze-dry the obtained peptides into peptide powder and store them at -80℃.

[0025] In summary, an OPA mixed reagent was prepared by adding 2.5 mL of 20% (w / w) sodium dodecyl sulfate, 40 mg of o-phthalaldehyde (dissolved in 1 mL of methanol), 25 mL of borax (0.1 M), and 100 μL of β-mercaptoethanol to 50 mL of deionized water. The OPA reagent was prepared fresh each time. 2 mL of OPA reagent was mixed with 100 μL of sample and incubated at room temperature in the dark for 2 min. The absorbance of the mixed solution was then measured at 340 nm using a microplate reader. The peptide content was calculated based on the trypsin peptone standard curve.

[0026] Assay of the antioxidant activity of 2.6 peptides 2.6.1 DPPH free radical scavenging activity Mix 100 μL of DPPH solution (0.2 mM dissolved in anhydrous ethanol) with an equal volume of sample (5 mg / mL). Incubate the mixture at room temperature in the dark for 1 h, then measure the absorbance at 517 nm. Calculate the DPPH radical scavenging activity using the following formula: ; Where A1 is the absorbance of the sample after mixing and reacting with DPPH solution, A2 is the absorbance of the sample after mixing and reacting with anhydrous ethanol, and A0 is the absorbance of the DPPH solution after mixing and reacting with anhydrous ethanol.

[0027] 2.6.2 ABTS free radical scavenging activity To prepare an ABTS stock solution, equal volumes of ABTS (7 mM) and potassium persulfate (2.5 mM) were mixed and incubated in the dark for 12–16 h. The solution was then diluted with deionized water to a absorbance of 0.70 ± 0.01 at 734 nm to obtain the ABTS working solution. 1 mL of sample and 1 mL of ABTS working solution were added to a centrifuge tube and incubated in the dark at room temperature for 15 min. The absorbance was then measured at 734 nm. The ABTS radical scavenging activity was calculated using the following formula: ; Where A1 is the absorbance after the sample is mixed with ABTS solution and reacted, and A0 is the absorbance after the deionized water is mixed with ABTS solution and reacted.

[0028] 2.6.3 Fe 2+Chelation ability Add 1 mL of sample to a mixed solution of 50 μL FeCl2 (2 mM) and 200 μL (5 mM) phenoxyazine. Vortex the mixture thoroughly and allow it to react at room temperature for 10 min. Then measure the absorbance of the mixed solution at 562 nm. Calculate the Fe2+ chelating capacity using the following formula: ; Where A1 is the absorbance of the sample after reacting with the mixed solution of FeCl2 and phenoxyazine, and A0 is the absorbance of deionized water after reacting with the mixed solution of FeCl2 and phenoxyazine.

[0029] 2.7 Determination of Free Amino Acids 0.2 g of cured duck was mixed with 8 mL of 4% (w / v) 5-sulfosalicylic acid and then extracted by sonication for 30 min. After centrifugation at 4 °C (12,000 × g, 30 min), the supernatant was filtered through a 0.22 μm filter membrane. The free amino acid content was then determined using an automated amino acid analyzer.

[0030] 2.8 nanoLC-MS / MS analysis The Evosepone nano-UPLC system was used with a mass spectrometer (timsTOFPro2) equipped with a nanoliter ion source. The HPLC system used a reversed-phase column (PePSepC18, 1.9m, 150m × 15cm). HPLC conditions: mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% formic acid acetonitrile solution. Mass spectrometry conditions: DDA data acquisition was performed in DDAAPASEF mode, with a scan range of 100-1700 m / z, a ramp time of 100 ms, and an Accutime of 100 ms. Peptide sequence analysis was performed using SpectroMine software (4.2.230428.52329; Biognosys AG).

[0031] 2.9 Bioinformatics Analysis Use Peptide Ranker ( http: / / distilldeep.ucd.ie / PeptideRanker / Peptides with potential biological activity were screened. Then, the BIOPEP-UWM database was used to screen for them. https: / / www.uwm.edu.pl / biochemia / index.php / pl / biopep Further predict the potential functional properties of peptides with a score greater than 0.5.

[0032] In addition, this study used ToxinPred ( https: / / webs.iiitd.edu.in / raghava / toxinpred / ) and AllergenFP ( https: / / ddg-pharmfac.net / AllergenFP / Assess peptide allergy and toxicity using Innovagen ( http: / / www.innovagen.com / proteomics-tools) and ProtParam https: / / web.expasy.org / protparam / The water solubility and stability of peptides were assessed using PepDraw. https: / / pepdraw.com / Tools are used to assess the physicochemical properties of peptides.

[0033] 2.10 Molecular docking The crystal structure of Keap1 (PDBID: 2FLU) was obtained from the PDB database (https: / / www.rcsb.org / ). The three-dimensional structure of the peptide was constructed using Chem3D software. Prior to docking, water molecules and the original ligand in the acceptor were removed using PyMOL 3.1.0 and Autodock Tools 1.5.7 (ADT), and hydrogen atoms were added. Molecular docking was performed using Autodock Vina 1.1.2. The interaction mode between the ligand and acceptor was analyzed using PyMOL 3.1.0 and LIGPLOT 2.3.0.

[0034] III. Results and Discussion 3.1. Physicochemical property analysis Table 1. Physicochemical properties of preserved duck during fermentation.

[0035] During the fermentation of the preserved duck, both moisture content and water activity (aw) continuously decreased in both treatment groups (Table 1). Notably, from the fourth day of fermentation until the end of fermentation, the moisture content of the inoculated group was significantly lower than that of the control group (P < 0.05). Similarly, at the end of fermentation, the water activity of the inoculated group was also significantly lower than that of the control group (P < 0.05). The difference may be due to the production of acidic substances by the bacterial strain's metabolism, which leads to protein denaturation and muscle contraction in the preserved duck, thereby reducing the water-holding capacity of the protein.

[0036] The pH changes of the duck meat during fermentation are shown in Table 1. At the beginning of fermentation (day 0), there was no significant change in pH between the control group and the inoculated group (P > 0.05). At the end of fermentation (day 10), the lowest pH values ​​were observed in the two groups, at 5.88 and 5.71, respectively (P < 0.05). Interestingly, from day 4 onwards, the pH value of the inoculated group was significantly lower than that of the control group (P < 0.05). This may be due to the large amount of acidic substances produced by the strain's metabolism, resulting in a lower pH value compared to the control group. Notably, the rate of pH decrease slowed down in the later stages of fermentation. This may be attributed to the production of alkaline ammonia from protein degradation.

[0037] 3.2. Bacterial Count Analysis Table 2. Changes in bacterial count during the fermentation process of preserved duck.

[0038]

[0039] Table 2 shows the changes in total bacterial count (TVC), lactic acid bacteria, and staphylococcus counts during the fermentation of preserved duck. As can be seen from Table 2, the total bacterial count in the inoculated groups was significantly higher than that in the control group during fermentation (P < 0.05). This indicates that L. curvatus D2 and S. simulans D12 can grow and reproduce well on preserved duck.

[0040] Lactic acid bacteria (LAB) play a crucial role in the fermentation process of preserved duck. Due to their acid-producing characteristics, they can inhibit the growth of putrefactive bacteria and pathogens, thus ensuring the safety and stability of the preserved duck. Overall, the number of LABs in both groups showed a trend of first increasing and then slightly decreasing. During the first 7 days of fermentation, the number of LABs increased rapidly. On day 7, the number of LABs in both groups reached their maximum values ​​of 6.17 lg CFU / g and 8.40 lg CFU / g, respectively. During the subsequent fermentation process, the number of LABs decreased. At the end of fermentation, the number of LABs in the two groups of preserved duck was 5.80 lg CFU / g and 8.13 lg CFU / g, respectively. The decrease in water activity or the reduction of nutrients in the preserved duck were the main factors contributing to the later decrease in the number of LABs. Furthermore, throughout the entire fermentation stage, the number of LABs in the inoculated group was significantly higher than that in the control group (P < 0.05).

[0041] The trends in Staphylococcus counts in the two groups of preserved ducks were similar to those in lactic acid bacteria. On day 0, the Staphylococcus counts in the control group and the inoculated group were 4.82 lg CFU / g and 6.87 lg CFU / g, respectively; on day 7, the counts reached their maximum values, at 6.14 lg CFU / g and 8.21 lg CFU / g, respectively; at the end of fermentation, the Staphylococcus counts in the two groups of preserved ducks were 5.57 lg CFU / g and 7.94 lg CFU / g, respectively. Furthermore, throughout the entire fermentation process, the counts in the control group were significantly lower than those in the inoculated group (P < 0.05). Notably, during most of the fermentation process, the number of lactic acid bacteria was higher than that of Staphylococcus. This may be due to the decrease in pH inhibiting the growth of Staphylococcus.

[0042] 3.3. Changes in protease activity

[0043] In fermented meat products, proteins are hydrolyzed by proteases to form peptides and amino acids, which regulate potential functional bioactive substances and flavor characteristics. The changes in protease activity between the control and inoculated groups during the fermentation process of cured duck are shown in the following figures. Figure 1As shown in Figure A, the enzyme activities in the control and inoculated groups were initially low, at 7.63 U / g and 7.48 U / g, respectively. From day 4 until the end of fermentation, the protease activity in the inoculated group was significantly higher than that in the control group (P < 0.05). This may be due to the secretion of exogenous proteases by the large-scale proliferation of microorganisms, which enhanced the protease activity of the duck. On day 7, the protease activities of both groups reached their peak, at 16.01 U / g and 19.66 U / g, respectively (P < 0.05). Interestingly, at the end of fermentation, the protease activities in both the control and inoculated groups began to decrease significantly (P < 0.05). This phenomenon may be due to changes in pH and water activity during the fermentation process of the duck. These data indicate that co-inoculation fermentation enhances the protease activity of the duck during fermentation.

[0044] 3.4. Protein hydrolysis index (PI) analysis The changes in PI values ​​of the control group and the inoculated group during the fermentation process of the preserved duck are as follows: Figure 1 As shown in Figure B, the PI values ​​of both the control and inoculated groups increased significantly with prolonged fermentation time (P < 0.05). On day 0 of fermentation, the PI value of the control group was 4.78%, increasing to 9.72% by the end of fermentation; on day 0 of fermentation, the PI value of the inoculated group was 4.74%, increasing to 11.57% by the end of fermentation. At the end of fermentation, the PI value of the inoculated group was significantly higher than that of the control group (P < 0.05).

[0045] The protein inoculant (PI) value directly reflects the degree of protein hydrolysis in salted duck during fermentation. Therefore, the higher PI value observed in the inoculated group at the end of fermentation is likely attributed to the increased protein hydrolysis by proteases secreted by the microorganisms. Furthermore, the inoculation with mixed strains produced a diverse range of proteases, further confirming that co-inoculation with *L. curvatus* D2 and *S. simulans* D12 promotes protein degradation in salted duck.

[0046] 3.5. Analysis of peptide content and antioxidant activity 3.5.1 Peptide content analysis Changes in peptide content during the fermentation process of cured duck, such as Figure 2As shown in Figure A, the peptide content in both the control and inoculated groups increased with increasing fermentation days. Initially, the peptide content in both groups was low, at 0.94% and 0.92%, respectively, with no significant difference (P > 0.05). From day 1 until the end of fermentation, the peptide content in the inoculated group was significantly higher than that in the control group (P < 0.05). At the end of fermentation, the peptide content in both groups reached its maximum, at 2.81% and 3.15%, respectively (P < 0.05). Microbial inoculation can effectively increase the peptide content in meat products. However, LAB inoculation did not significantly increase the peptide concentration in fermented sausages. This phenomenon may be due to the increased protease diversity in duck meat resulting from the co-inoculation method. These results indicate that co-inoculation with Lactobacillus curvatus and Staphylococcus simulans can degrade muscle proteins into small peptides. Of course, whether these crude peptides possess antioxidant activity requires further analysis.

[0047] 3.5.2 Antioxidant Activity Analysis Changes in the antioxidant activity of crude peptides extracted from cured duck during fermentation, as follows: Figure 2 As shown in B, 2C, and 2D, the DPPH radical scavenging capacity of both the control and inoculated groups increased from the beginning to the end of fermentation. At the end of fermentation, the DPPH radical scavenging capacity of both groups reached its maximum, at 35.90% and 44.91%, respectively (P < 0.05). Furthermore, the increase in DPPH radical scavenging capacity in the inoculated group was not significant within 7-10 days (P > 0.05). In this study, the ABTS radical scavenging capacity of the crude peptide increased significantly with fermentation time (P < 0.05), and its trend was similar to that of the peptide content. In addition, the Fe content of the peptide... 2+ The chelating capacity generally showed a trend of first increasing and then decreasing in both groups. On day 7, the highest values ​​were reached in both groups, at 16.53% and 18.35%, respectively. At the end of fermentation, the Fe content of the peptides... 2+ The chelating ability decreased. This may be due to changes in the peptide structure leading to alterations in the active sites. These data reveal that inoculation with Lactobacillus curvatus and Staphylococcus simulans can significantly enhance the antioxidant activity of peptides during the fermentation of preserved duck and promote the formation of potential antioxidant peptides.

[0048] 3.6. Analysis of Free Amino Acids Table 3. Composition and content of free amino acids in salted duck (mg / 100g)

[0049] Proteins in cured duck are broken down into free amino acids by proteases. Table 3 shows the free amino acid content of cured duck on day 0 and at the end of fermentation. Compared with day 0, the content of various amino acids increased to varying degrees in both the control group and the inoculated group at the end of fermentation, and the total free amino acid content showed a significant difference (P < 0.05). The total free amino acid content in the inoculated group was 1364.06 mg / 100g, significantly higher than that in the control group (1107.41 mg / 100g, P < 0.05), indicating that inoculation with L. curvatus D2 and S. simulans D12 can significantly increase the free amino acid content in cured duck. In addition, the content of some hydrophobic amino acids (such as alanine, valine, isoleucine, leucine, and phenylalanine) in the inoculated group was significantly higher than that in the control group (P < 0.05). Hydrophobic amino acids in polypeptide chains have a positive effect on antioxidant activity. Therefore, this result shows that the presence of hydrophobic amino acids affects the antioxidant activity of cured duck.

[0050] 3.7. Peptidomics Analysis 3.7.1 Mass spectrometry identification data analysis To investigate the differences in protein and peptide composition between natural fermentation and inoculated fermentation, peptide profiling was performed using LC-MS / MS. Changes in the number of peptides can reflect the degree of protein degradation into smaller peptide molecules. 3348 and 4022 peptides were identified in the control and inoculated groups, respectively. Figure 3 A) and 275 and 309 proteins ( Figure 3 (B) These findings indicate that inoculation and fermentation not only increased peptide concentration but also altered peptide sequences. Furthermore, the diversity of peptide sequences and parental proteins is crucial for assessing differences in peptide release across different groups. Figure 3 As shown in Figure A, a total of 3808 unique peptides were found in the two groups of samples, with 2241 and 1567 respectively. The observation of a large number of unique peptides in the inoculated group indicates different protein degradation patterns before and after inoculation and fermentation. This difference may be due to changes in enzyme cleavage sites and synergistic effects between exogenous and endogenous proteases. Furthermore, different unique proteins were also observed in the two groups, with 93 and 59 proteins respectively. These results suggest that microbial metabolic activity may alter protein degradation pathways in the samples, leading to differences in the final peptide sequences.

[0051] To further investigate the effect of inoculation and fermentation on peptide profiles, the length and molecular weight distribution of the identified peptides in the two groups of samples were analyzed. Figure 3As shown in C and 3D, the peptide length distribution in both groups of samples was similar, mainly concentrated in the 7-21 amino acid range. Furthermore, the molecular weights of the identified peptides were primarily in the 3000 Da range. Notably, for peptides with molecular weights less than 1.5 kDa or between 1.5 and 3 kDa, their numbers were significantly increased in the inoculated groups compared to the control group. It has been reported that low molecular weight peptides possess stronger antioxidant activity, which may be related to their ability to expose larger active sites. This result confirms that the crude peptides in the inoculated groups exhibited higher antioxidant activity.

[0052] Furthermore, studies have shown that hydrophobic amino acids (Ala, Val, Leu, Pro, Met, Phe, Ile) present at the N-terminus or C-terminus of peptide chains can significantly enhance their antioxidant activity. As shown in the figure, the proportion of peptides containing hydrophobic amino acid residues at the N-terminus and C-terminus in the control group was 75.09%, while the proportion in the inoculated group was 74.66%, with the two groups showing similar percentages. However, analysis of the number of peptides revealed that 1906 peptides in the inoculated group had hydrophobic amino acids at their N-terminus, and 1097 peptides had hydrophobic amino acids at their C-terminus. In contrast, 1577 and 937 peptides in the control group contained the corresponding amino acid residues. This can partially explain the relatively higher antioxidant activity of the crude peptides in the inoculated group.

[0053] 3.7.2 Parental protein analysis for peptide identification To investigate the degradation of proteins in preserved duck by inoculation and fermentation, the parental protein source of the identified peptides was further analyzed. For example... Figure 4 As shown in the figure. In the control group, peptides mainly originated from M-type creatine kinase (13.20%), troponin T3 (12.01%), myosin heavy chain (8.09%), myoglobin (6.15%), and troponin I (5.94%), accounting for a total of 45.40%. In the inoculated group, peptides mainly originated from troponin T3 (8.95%), myosin heavy chain (8.28%), M-type creatine kinase (7.71%), myoglobin (6.32%), phosphoglycerate kinase (4.72%), troponin I (4.57%), and ATP synthase β subunit (4.20%), accounting for a total of 44.75%. Observation of the parental protein distribution of the identified peptides revealed that M-type creatine kinase, troponin T3, myoglobin, and myosin heavy chain were the main sources of the identified peptides in both groups. In addition, a significant difference in the proportion of parental proteins was observed between the two groups, indicating that inoculation fermentation altered the protein degradation behavior. This result is significant in... Figure 3 This is also reflected in B.

[0054] Hierarchical cluster analysis was used to further investigate the effect of inoculation fermentation on peptide release from parental proteins. This study focused on identifying the 30 parental proteins with the highest peptide sources. Figure 4As shown in Figure C, the different colors on the heatmap, from blue to red, represent the relative amounts of peptides released from the corresponding parental proteins. Overall, the inoculated group had the highest relative amounts of the identified peptides derived from the corresponding parental proteins. Furthermore, compared to the control group, the inoculated group showed an increased relative amount of peptides produced by A0A8B9TVN7 (myosin heavy chain), A0A8B9UNS6 (myoglobin), A0A8B9T8Z7 (phosphoglycerate kinase), A0A8B9VII4 (glyceraldehyde-3-phosphate dehydrogenase), and A0A493T6U6 (enolase), indicating that the microorganisms promoted the degradation of these proteins. This difference may be due to the synergistic effect of endogenous and exogenous enzymes. The endogenous enzymes initially create cleavage sites, which are then further recognized by the exogenous proteases from the microorganisms, enabling them to bind and catalyze effectively.

[0055] In summary, the qualitative and quantitative changes in parental protein sources reflect the degree of protein degradation in the samples. This further confirms that inoculation fermentation has different effects on different types of proteins, leading to differences in the peptide sequences of protein degradation products. Of course, these differences also have a significant impact on the release of bioactive peptides and their biological functions.

[0056] 3.7.3 GO Analysis and KEGG Analysis To analyze the biological function of differentially expressed peptide-derived parental proteins, GO functional annotation analysis was performed. For example... Figure 6 As shown in Figure A, biological processes include small molecule metabolism (GO:0044281), precursor metabolite and energy production (GO:0006091), carboxylic acid metabolism (GO:0019752), translation (GO:0006412), and peptide biosynthesis (GO:0043043). These proteins may play key roles in cellular energy metabolism and protein metabolism. From the perspective of cellular composition, differentially expressed proteins are mainly concentrated in intracellular anatomical structures (GO:0005622), cytoplasm (GO:0005737), protein complexes (GO:0032991), mitochondria (GO:0005739), and myofibrils (GO:0030016). These annotations are closely related to intracellular metabolism and signal transduction. In terms of molecular function, cytoskeletal protein binding (GO:0008092) involved the most differentially expressed proteins, followed by actin binding (GO:0003779) and ribosome structural components (GO:0003735). These annotations suggest that parental proteins may play roles in maintaining cell morphology and intracellular transport. Furthermore, KEGG metabolic pathway analysis was used to further investigate the pathways involving parental proteins in differentially expressed peptides. Figure 6As shown in B, these metabolic pathways are mainly concentrated in six categories: metabolism, genetic information processing, environmental information processing, cellular processes, organismal systems, and human diseases. Among them, the five most significantly enriched metabolic pathways are carbon metabolism, amino acid biosynthesis, the citric acid cycle (TCA cycle), the cytoskeleton in muscle cells, and ribosomes. These results indicate that during the fermentation and maturation of preserved duck, exogenous and endogenous enzymes work together to break down muscle proteins, thereby producing small peptides and amino acids.

[0057] 3.8. Analysis of peptide functional properties Bioactive peptides, typically composed of 2-20 amino acid residues, are specific sequences derived from food proteins. They are usually hidden within the complex structure of proteins and only exhibit specific biological activities after cleavage. Therefore, to determine whether changes in the number of bioactive peptides induced by inoculation fermentation are the primary cause of increased antioxidant activity in crude peptides, the online tool PeptideRanker was used to predict and identify the bioactivity of peptides. In this study, peptides predicted to exceed a threshold of 0.5 were considered potentially bioactive peptides. Figure 5 A illustrates the correlation between bioactive peptides in the two groups of samples. The number of potentially bioactive peptides observed in the control group and the inoculated group were 306 and 446, respectively. Furthermore, the predicted percentages of bioactive peptides in the total peptides were 11.09% and 9.14% in the inoculated group and the control group, respectively. These results further indicate that *L. curvatus* D2 and *S. simulans* D12 promote the formation of bioactive peptides in salted duck.

[0058] To further investigate the specific functions of these bioactive peptides, BIOPEP-UWM was used for prediction. Figure 5 Figures B and 5C illustrate the functional distribution of bioactive peptides in the two groups of samples. Compared to the control group, the inoculated group exhibited higher levels of DPPIII inhibitors, ACE inhibitory peptides, antioxidant peptides, DPPIV inhibitors, and enkephalinase inhibitors. Specifically, potential antioxidant peptides accounted for 7.44% of the total identified peptides in the control group, while they accounted for 8.85% in the inoculated group. Furthermore, the number of potential antioxidant peptides, ACE inhibitory peptides, and DPPIII inhibitory peptides in the co-inoculated fermentation group of *L. curvatus* D2 and *S. simulans* D12 was significantly higher than that in the control group. Analysis of the bioactive peptide types in both groups revealed that the inoculated group contained a greater variety of functional peptides and exhibited richer functionalities than the control group.

[0059] 3.9 Screening of antioxidant peptides Table 4 Physicochemical properties of the identified peptides

[0060] To further explore antioxidant peptides in the inoculated fermentation group, this study used computer methods for further screening based on the PeptideRanker and BIOPEP-UWM predictions of potential antioxidant peptides. Traditional methods for screening antioxidant peptides have many limitations; therefore, computer-based virtual screening has been widely used. This study screened peptides with predicted antioxidant activity in the inoculated group based on molecular weight (less than 1500 Da), safety, stability, water solubility, and sensitization prediction. Table 4 shows the sequences of 13 novel peptides after screening and their related prediction information. The results showed that these 13 peptides had hydrophobicity between 12.47 and 25.93 kcal / mol, pI values ​​between 4.01 and 1.71, and all exhibited good stability and water solubility, and were non-toxic and non-sensitizing. Therefore, these 13 peptides are potential antioxidant peptides. Furthermore, these peptides were further analyzed using molecular docking.

[0061] 3.10 Analysis and docking Table 5. Binding energy of peptide sequences to Keap1 protein

[0062] The Keap1-Nrf2 signaling pathway is a major mechanism regulating cellular antioxidant responses. Studies have shown that antioxidant peptides can occupy Keap1-Nrf2 binding sites, thereby inhibiting the interaction between Keap1 and Nrf2 and activating this antioxidant pathway. This study aimed to screen for antioxidant peptides with binding potential using molecular docking technology and further explore the interaction between target peptides and the Keap1 protein receptor. The binding energies required for the docking of 13 peptides with Keap1 are shown in Table 5. Lower binding energies indicate easier interaction between the peptide and ligand. The binding energies of GIRLGLDPKL (GI10), PAPAAKAGASTGRIV (PV15), and PEKPPTIDWA (PA10) with Keap1 are -7.1 kcal / mol, -8.2 kcal / mol, and -7.8 kcal / mol, respectively. They rank among the top three of the 13 peptides. Therefore, GI10, PV15, and PA10 may possess strong antioxidant activity, and further analysis of their docking details is needed.

[0063] Table 6. Binding sites and binding forces of the three peptides with Keap1 protein

[0064] The docking results of GI10, PV15, and PA10 with Keap1 are as follows: Figure 7As shown in Table 6, the interactions between GI10 and Keap1 are as follows. Docking results indicate that GI10 forms hydrogen bonds with amino acid residues Val463, Val369, Val608, Val420, and Arg326. Furthermore, this peptide forms hydrophobic interactions with Keap1 residues Val604, Gly367, Val606, Ala366, Val465, Ile559, Gly417, Cys368, Gly464, Cys513, Ala466, Val467, Val514, His516, Leu515, Gln563, Gly564, Val561, Ala607, Thr560, Leu557, and Gly558. PV15 forms hydrogen bonds with amino acid residues of Keap1 at Arg326, Val369, Gly367, Val606, Ala510, Ser363, Ile416, and Arg380, and forms hydrophobic interactions with amino acid residues Val561, Val420, Val467, Val514, Val465, Ile559, Val463, Gly462, Gly417, Gly509, Gly464, Val418, Ala556, Gly603, Arg415, Leu557, Tyr334, Phe577, Tyr572, Gly364, Ser602, and Ala366. PA10 forms hydrogen bonds with amino acid residues of Keap1 at Val514, Val369, Val467, Val608, Val561, and Thr560. Furthermore, PA10 forms hydrophobic interactions with Leu515, His516, Leu468, Cys513, Ala466, Val420, Val418, Gly367, Val465, Val512, Gly419, Leu557, Ile559, Gly558, Val606, Cys368, Arg326, and Ala60 of Keap1. These results demonstrate that hydrogen bonding and hydrophobic interactions are the main forces driving peptide binding to the Keap1 receptor. Therefore, GI10, PV15, and PA10 may exert their antioxidant activity by activating the Keap1-Nrf2 pathway.

[0065] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A type of antioxidant peptide from preserved duck, characterized in that, Its amino acid sequence is at least one of GIRLGLDPKL, PAPAAKAGASTGRIV, and PEKPPTIDWA, as shown in SEQ ID NO: 1-3.

2. A method for preparing antioxidant peptides from preserved duck, characterized in that, Includes the following steps: Step S1. Apply 6wt% salt evenly to the surface and abdominal cavity of the slaughtered duck and let it stand at 4℃ for 24 hours. After dry curing, further marinate the duck in saturated brine at 4℃ for 12 hours and place it in a well-ventilated environment to drain the surface moisture. Step S2. Inoculate Lactobacillus curvature and Staphylococcus mimicus into liquid culture medium, incubate at 37°C for 24 hours, and then subculture for 12 hours. After centrifugation to collect the bacterial cells, the cells were washed three times with 0.85% sterile physiological saline and resuspended to obtain a concentration of 10. 7 A bacterial suspension of cfu / mL was stored at 4°C for later use. Step S3. Mix the above bacterial suspension with Lactobacillus curvature and Staphylococcus mimicus in a volume ratio of 1:1, and inoculate it evenly onto the surface and interior of the duck treated in Step 1 by spraying and injection; then air dry it at 25°C and 65% relative humidity for 1 day, and then air dry it at 18 to 20°C and 65% relative humidity for 9 days to obtain the fermented and matured duck product. Step S4. Take the duck muscle and mix it with hydrochloric acid solution, homogenize it under ice bath conditions, then let it stand at 4°C for 2 hours, filter and centrifuge to obtain the supernatant to obtain crude duck polypeptide; obtain crude peptide solution by ultrafiltration, and freeze dry it under vacuum to obtain duck antioxidant peptide powder.

3. The application of the duck antioxidant peptide according to claim 1 or 2 in the preparation of food preservatives.

4. The application of the duck antioxidant peptide according to claim 1 or 2 in the preparation of functional foods or nutritional supplements.

5. The use of the duck antioxidant peptide according to claim 1 or 2 in the preparation of antioxidants.

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