A polypeptide NPAAKMSPKPATP and its application in biological preservatives

Abstract

The present invention discloses a polypeptide NPAAKMSPKPATP and its use in a biological preservative, comprising the following steps: S1, preparation of a Paenibacillus Provence PP-2 metabolite: after solidification of LB solid medium, Paenibacillus Provence PP-2 is inoculated and streaked, then placed in a constant temperature incubator and incubated upside down for 48 hours, a single colony is picked and inoculated into LB liquid medium, incubated in a sterile shaker for 24 hours, and then inoculated into LB liquid medium at a 1% addition rate and cultured for 7 days. Subsequently, the mixture is centrifuged at 8000 rpm and 4°C for 10 minutes, the precipitate is discarded, and the supernatant is filtered and sterilized with a 0.22 μm filter membrane to obtain a Paenibacillus Provence PP-2 metabolite; P. The present invention uses a polypeptide prepared from the Paenibacillus Provence PP-2 metabolite and combines it with proanthocyanidins and agar oligosaccharides to prepare a biological preservative. The biological preservative has good preservative and antibacterial effects, and can overcome the shortcomings of traditional preservation methods.

Description

Technical Field

[0001] The present invention relates to the technical field of preparation of biological preservatives, in particular to a polypeptide NPAAKMSPKPATP and application thereof in biological preservatives. Background Art

[0002] Due to their high moisture content, fresh aquatic products, typically stored at 4°C, are susceptible to endogenous enzymes and microorganisms, leading to spoilage and affecting their edible value. Traditional preservation methods have significant drawbacks in addressing this issue. For example, chemical preservatives can leave harmful residues in food, posing potential food safety risks. Physical preservation methods are also costly and have limitations in their effectiveness, making them difficult to meet the demand for long-term, efficient preservation. However, biological preservatives, due to their natural, environmentally friendly, and safe properties, are becoming a growing trend in the food preservation field.

[0003] Currently, there has been no systematic report on the role of Paenibacillus Provence PP-2 metabolites in aquatic product preservation, leaving significant research gaps and room for exploration. In-depth research on Paenibacillus Provence PP-2 metabolites opens up broad prospects for the development of new, highly effective biopreservatives, with high research value and application potential. Biopreservatives, due to their natural, green, and safe properties, have gradually become a research hotspot in the field of aquatic product preservation. Paenibacillus Provence PP-2 metabolites, as a potential source of natural peptides, combined with proanthocyanidins and agar-oligosaccharides, are expected to overcome the shortcomings of traditional preservation methods and provide new directions for innovation in aquatic product preservation technology.

[0004] Furthermore, proanthocyanidins are potent natural antioxidants that effectively scavenge free radicals and slow the oxidation process, which helps extend the shelf life of fish and preserve its color, flavor, and nutritional content. Agar-oligosaccharides, as natural, water-soluble polysaccharides, form gels that enhance the fish's water retention capacity and reduce water loss, thereby preventing the fish from drying out and deteriorating in texture. Proanthocyanidins also possess antimicrobial properties, inhibiting the growth of microorganisms on and within the fish, helping to reduce the risk of food spoilage. The viscosity of agar-oligosaccharides forms a protective film, further blocking microbial invasion.

[0005] In order to solve the above technical problems, the present invention provides a polypeptide NPAAKMSPKPATP derived from the metabolite of Paenibacillus provence PP-2 and its application in preparing a biological preservative. Summary of the Invention

[0006] The invention provides a polypeptide NPAAKMSPKPATP and application thereof in a biological preservative, which solves the problems in the background technology.

[0007] In order to achieve the above object, the present invention adopts the following technical solutions:

[0008] A polypeptide NPAAKMSPKPATP comprises the following steps:

[0009] S1. Preparation of Paenibacillus Provence PP-2 metabolites: After solidification, Paenibacillus Provence PP-2 was streaked onto solid LB medium. The medium was then incubated upside down in a constant-temperature incubator for 48 hours. A single colony was picked and inoculated into LB liquid medium and incubated in a sterile shaker for 24 hours. The culture was then added to LB liquid medium at a 1% concentration and cultured for 7 days. The supernatant was then centrifuged at 8000 rpm and 4°C for 10 minutes, and the pellet was discarded. The supernatant was sterilized by filtration through a 0.22 μm filter to obtain Paenibacillus Provence PP-2 metabolites.

[0010] S2. Isolating and purifying the polypeptide NPAAKMSPKPATP from the metabolites in step S1: Using gel column chromatography to further screen the antibacterial components of the Paenibacillus Provence PP-2 metabolites to obtain peptide fragments, and then identifying and analyzing the peptide sequences. After analysis, the polypeptide NPAAKMSPKPATP is synthesized.

[0011] Furthermore, in step S2, the peptide sequence is identified by LC-MS method, and the mobile phase A of the peptide sequence is 0.1% formic acid and 2% acetonitrile; the mobile phase B is 0.1% formic acid and 80% acetonitrile.

[0012] Furthermore, in step S2, the peptide sequence is analyzed de novo using a Q-Exactive HF-X mass spectrometer in a data-dependent acquisition mode for mass spectrometry analysis.

[0013] Furthermore, in step S2, the polypeptide NPAAKMSPKPATP is synthesized using the Fmoc solid phase synthesis method.

[0014] Another object of the present invention is to provide an application of the polypeptide NPAAKMSPKPATP in a biological preservative.

[0015] Furthermore, a method for preparing a biological preservative containing the polypeptide NPAAKMSPKPATP is as follows: polypeptide NPAAKMSPKPATP powder is dissolved in sterile distilled water, and proanthocyanidins and agar oligosaccharide are added and mixed uniformly to obtain a composite biological preservative.

[0016] Furthermore, the concentration of the polypeptide NPAAKMSPKPATP powder after being dissolved in sterile distilled water is 180 mg / mL.

[0017] Furthermore, the mass concentration of the proanthocyanidins is 0.2% w / v.

[0018] Furthermore, the mass concentration of the agar oligosaccharide is 0.5% w / v.

[0019] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0020] The present invention uses a polypeptide prepared from the metabolite of Paenibacillus Provence PP-2, and combines it with proanthocyanidins and agar oligosaccharides to prepare a biological preservative. The biological preservative has good preservative and antibacterial effects and can overcome the shortcomings of traditional preservation methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the mass spectrum of the peptide NPAAKMSPKPATP;

[0022] Figure 2 The bacterial survival rate curve under different treatment conditions;

[0023] Figure 3 Transmission electron micrographs of Stenotrophomonas microacidophilus under different treatment conditions;

[0024] Figure 4 The figure shows the effect of different conditions on the cell activity of Stenotrophomonas microacidiophylla;

[0025] Figure 5 This is a graph showing the changes in the SDS-PAGE electrophoresis results of myofibrillar protein over time;

[0026] Figure 6 This is a graph showing the changes in the microstructure of myofibrillar protein over time;

[0027] Figure 7 This figure shows the effect of composite biological preservatives on the microstructure of protein gel. DETAILED DESCRIPTION

[0028] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.

[0029] The preparation process of a polypeptide NPAAKMSPKPATP is as follows:

[0030] Step 1, preparation of Paenibacillus Provence PP-2 metabolites and separation and purification of the polypeptide NPAAKMSPKPATP:

[0031] Paenibacillus provencensis PP-2 was isolated from sturgeon caviar produced by Xunlong Technology Co., Ltd. in Quzhou, Zhejiang Province. It is deposited with the China Center for Type Culture Collection under the accession number CCTCC No. M 20211109. The deposit information and details of this strain are described in Chinese invention patent CN 114134085A. CN 114134085A discloses its use in inhibiting the formation of nitrosamines in food. Paenibacillus provencensis PP-2 was inoculated and streaked onto LB solid medium after solidification, then incubated in an inverted incubator for 48 hours. A single colony was picked and inoculated into LB liquid medium, incubated in a sterile shaker for 24 hours, and then added to LB liquid medium at a 1% concentration for 7 days. The mixture was then centrifuged at 8000 rpm and 4°C for 10 min, and the supernatant was discarded and sterilized by filtration through a 0.22 μm filter to obtain the Paenibacillus provence PP-2 metabolite. Antibacterial components of the Paenibacillus provence PP-2 metabolite were further screened using gel column chromatography, resulting in the identification of the peptide with the best antibacterial activity. The peptide sequence was identified by LC-MS. Mobile phase A consisted of 0.1% formic acid and 2% acetonitrile, and mobile phase B consisted of 0.1% formic acid and 80% acetonitrile. De novo peptide sequence analysis was performed using a Q-Exactive HF-X mass spectrometer in data-dependent acquisition mode. NPAAKMSPKPATP (Asp-Pro-Ala-Ala-Lys-Met-Ser-Pro-Lys-Pro-Ala-Thr-Pro) was identified as the most abundant peptide, indicating that this peptide is closely related to the antibacterial activity of the Paenibacillus provence PP-2 metabolite. The peptides were synthesized using the Fmoc solid-phase synthesis method.

[0032] The application of a polypeptide NPAAKMSPKPATP in biological preservatives is studied as follows:

[0033] Stenotrophomonas acidaminiphila was used as the experimental strain, and the antibacterial activity of PP-2 metabolites was determined by microdilution method. Nisin and polypeptide NPAAKMSPKPATP were added to the activated fermentation broth, respectively, to make the metabolite concentrations of 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, 150 mg / mL, 180 mg / mL, 200 mg / mL, and 250 mg / mL. After mixing, 200 μL was added to a 96-well plate. The culture medium containing the fermentation broth of Stenotrophomonas acidaminiphila at the same concentration was used as the blank control group. The culture was incubated at 30°C in a sterile shaker for 12 h, and the OD value was measured.600 size.

[0034] Antibacterial activity against Stenotrophomonas microacidophilus under different treatment conditions. As the concentration of the peptide NPAAKMSPKPATP increased, the inhibition rate initially increased and then stabilized. At a concentration of 180 mg / mL, the peptide NPAAKMSPKPATP achieved an inhibition rate of 73.11±1.77% against Stenotrophomonas microacidophilus. At concentrations above 180 mg / mL, the inhibition rate remained unchanged. Therefore, the minimum inhibitory concentration (MIC) of the peptide NPAAKMSPKPATP against Stenotrophomonas microacidophilus was determined to be 180 mg / mL. The minimum inhibitory concentration (MIC) of Nisin in the control group was 100 mg / mL.

[0035] like Figure 2 As shown, transmission electron microscopy was used to observe the morphological changes of S. microacidophilus cells. Samples were removed after 6 hours of culture and centrifuged at 8000 rpm and 4°C for 10 minutes. The supernatant was discarded and the pellet was retained. Sterile PBS was added to the pellet, centrifuged, and the supernatant was discarded. This was repeated three times. 2.5% (v:v) glutaraldehyde fixative was added to the pellet and fixed overnight at 4°C. A 10 μL droplet of the bacterial solution was placed on a copper grid. After drying, the sample was washed with sterile PBS and dried again. Finally, the treated sample was placed under a transmission electron microscope to observe the morphological changes of S-1 cells.

[0036] Transmission electron microscopy revealed the cell morphology of the blank control group, Nisin group, and peptide NPAAKMSPKPATP group treated with the bacteria. The cells in the blank control group were plump and morphologically intact. The cells in the Nisin group were relatively intact, showing no significant changes. However, the cell membranes in the peptide NPAAKMSPKPATP group dissolved, severely damaging the bacteria. Cavities formed in the cells, causing leakage of contents and the inability to maintain normal cell growth and metabolism, leading to cell death. This result confirms that the peptide NPAAKMSPKPATP can inhibit bacteria by disrupting cell membrane integrity.

[0037] like Figure 3As shown, propidium iodide (PI) single staining was used to examine the lethality of the peptide NPAAKMSPKPATP against Stenotrophomonas microacidophilus. Samples were removed after 6 hours of incubation and centrifuged at 8000 rpm and 4°C for 10 minutes. The supernatant was discarded and the pellet was washed three times with sterile PBS. The pellet was resuspended in sterile PBS and analyzed using the PI Cell Viability and Cytotoxicity Assay Kit. To a sterile centrifuge tube, 100 μL of bacterial suspension and 30 μL of PI stain were added. The cells were incubated in a dark incubator for 30 minutes. The cells were centrifuged at 5000 rpm and 4°C for 10 minutes. The supernatant was discarded and the pellet was washed three times with sterile PBS and resuspended in sterile PBS. 10 μL of the resuspended bacterial suspension was transferred to a glass slide, mounted with anti-fluorescence decay medium, and observed under a motorized fluorescence microscope.

[0038] Propidium iodide (PI) is a fluorescent dye that intercalates into DNA. When the cell membrane is intact, PI is blocked by the cell membrane and cannot enter the cell. However, once the cell membrane structure is disrupted and its integrity is compromised, PI can enter the cell and bind to DNA, generating a fluorescent signal. Therefore, after PI staining, monitoring changes in fluorescence intensity can be used to assess the presence and extent of cell membrane damage. As shown in the figure, the microacidophilic Stenotrophomonas bacteria treated with Nisin exhibited less red fluorescence, while the majority of cells exhibited red fluorescence after treatment with the peptide NPAAKMSPKPATP. This suggests that the peptide NPAAKMSPKPATP can alter cell membrane permeability, leading to cell death and allowing PI to enter the cell, bind to DNA, and generate red fluorescence.

[0039] The method of preparing a biological preservative containing the polypeptide NPAAKMSPKPATP is as follows:

[0040] The polypeptide NPAAKMSPKPATP powder was dissolved in sterile distilled water at a concentration of 180 mg / mL, and 0.2% (w / v) proanthocyanidins and 0.5% (w / v) agar oligosaccharide were added and mixed evenly to obtain a composite biological preservative.

[0041] Table 1 Solution formula ratio of each experimental group

[0042]

[0043] The following experiments were conducted on the biological preservative containing the polypeptide NPAAKMSPKPATP:

[0044] Verification of preservation effect:

[0045] Fresh tilapia (500 ± 30 g, 32 ± 0.5 cm in length, and 14 ± 0.5 cm in width) were used. Sterile distilled water served as a blank control. A certain number of tilapia fillets were placed in each solution (mass ratio of fillet to solution was 1:10) and immersed at 4°C for 30 min. After immersion, the samples were drained of surface moisture using a sterile operating table, vacuum-packed in sterile vacuum bags, and stored at 4°C. Samples were collected on days 0, 1, 3, 5, and 9 for measurement of various parameters.

[0046] The fish fillets were removed and minced. 10.00 g of fish meat sample was accurately weighed and added to 40 mL of pre-chilled protein buffer (10 mmol / L Na₃PO₄, 0.1 mol / L NaCl, 2 mmol / L MgCl₂, 1 mmol / L EGTA, pH 7.0). Homogenize at 10,000 rpm for 1 min, stopping three times. The sample was then centrifuged at 5,000 rpm at 4°C for 10 min. The supernatant was discarded, and the pellet was resuspended in 40 mL of buffer. The extraction and centrifugation steps were repeated three times. Finally, the pellet was resuspended in 40 mL of 0.1 mol / L NaCl solution and homogenized under the same conditions to remove connective tissue. After centrifugation at 5,000 rpm at 4°C for 10 min, the pellet was collected to obtain purified myofibrillar protein.

[0047] Add 10 times the volume of 15 mmol / L PIPES buffer (containing 0.6 mol / L NaCl, pH 6.25) to the precipitate, homogenize at 10,000 rpm for 1 min, let it stand at 4°C for 30 min, filter through two layers of gauze, centrifuge and store the supernatant at 4°C for subsequent determination.

[0048] Experiment 1: Effects on the Total Colony Count in Tilapia Fillets

[0049] Microbial growth is one of the important factors that lead to spoilage of aquatic products, so the total number of colonies can be used to evaluate the freshness of aquatic products. According to national health standards, the total number of colonies in first-grade fresh meat should be ≤10 6 CFU / g, the total colony count of secondary fresh meat ranges from 10 6 CFU / g-10 8 CFU / g, when the total number of colonies reaches 10 8 If the CFU / g is above, it is judged as rotten meat.

[0050] The total colony counts of refrigerated tilapia fillets under different treatments are shown in the table. The total colony counts increased with storage time, showing an upward trend. On day 0, the initial colony counts in experimental groups 1, 2, and 3 were 3.88 ± 0.31 log CFU / g, 3.79 ± 0.14 log CFU / g, and 3.84 ± 0.05 log CFU / g, respectively, all at the level of first-grade fresh meat. On day 5 of storage, the colony counts in experimental groups 1 and 2 were 7.30 ± 0.56 log CFU / g and 6.81 ± 0.01 log CFU / g, respectively, both at the level of second-grade fresh meat. The colony count in experimental group 2 was 5.72 ± 0.15 log CFU / g, lower than the other two groups and at the level of first-grade fresh meat. On day 9, the colony count in the fillets of experimental group 2 reached 8.39 ± 0.05 log CFU / g, indicating spoilage. By comparison, experimental group 2 showed significant advantages in inhibiting the growth of total bacterial counts in tilapia fillets and extending their shelf life. The peptide NPAAKMSPKPATP can inhibit the growth and reproduction of microorganisms, delaying the deterioration of the quality of refrigerated tilapia fillets and effectively extending their shelf life.

[0051]

[0052] Experiment 2 - Effects of Composite Biopreservatives on Myofibrillar Protein Degradation Bands in Tilapia Fillets

[0053] Tilapia dorsal meat samples (3 × 3 × 3 mm) were cut from each group and fixed in 10% formalin for 24 hours to prevent tissue decay and preserve tissue morphology. After fixation, the tissue samples were dehydrated using ethanol in increasing concentrations (50%, 60%, 70%, 80%, 90%, and 100%) for approximately 1 hour at each concentration to remove the fixative and render the tissue anhydrous. After dehydration, the tissue samples were embedded and sectioned. The cut surface of the tissue sample was first immersed in paraffin solution, avoiding the formation of bubbles. After infiltration, the sample was poured into a mold and placed on a cooling plate to solidify the paraffin. After solidification, the sample was removed and sliced ​​using a tissue slicer. Thin slices approximately 5 μm thick were then floated in a 40°C water bath, flattened, and mounted on glass slides for observation and analysis of tissue structure. The samples were then stained with hematoxylin and eosin. After each treatment, the samples were observed and photographed using a light microscope.

[0054] It can be seen that during the early storage period, the myofibrillar structure was compact and neatly arranged, with no obvious gaps. By the middle storage period, the myofibrillar structure in each group became smaller, with clear boundaries and the beginning of gaps. By the late storage period, larger extracellular spacing and irregular pores were observed in the myofibrillar structure of experimental group 1, while experimental groups 2 and 3 inhibited structural changes to varying degrees. Compared with experimental group 3, experimental group 2 maintained a smaller and fewer gaps in the structure, effectively reducing structural collapse during cold storage. This result also reflects that the peptide NPAAKMSPKPATP can effectively inhibit the degradation of myofibrillar proteins.

[0055] Experiment 3 - Observation of the Microstructure of Myofibrillar Protein Gel in Tilapia Fillets

[0056] The treated myofibrillar protein was adjusted to 40 mg / mL with 15 mmol / L PIPES buffer (containing 0.6 mol / L NaCl, pH 6.25) and placed in a glass bottle. It was heated from 25°C to 80°C at a rate of 1°C / min and kept heating for 30 minutes. It was placed in a cold water bath for cooling and then placed in a 4°C refrigerator overnight for 12 hours before use.

[0057] The gel samples to be tested were fixed with 2.5% glutaraldehyde (0.1 M, pH 7.2) at 4°C for 24 h. The samples were then washed three times with PBS (0.1 M, pH 6.8) for 10 min each. The gel samples were then dehydrated three times with 40%, 50%, 60%, 70%, 80%, 90%, and 100% ethanol for 10 min each. The samples were then dried and gold-sprayed. The microstructure of the samples was observed using a scanning electron microscope at 500x magnification.

[0058] The microstructure of protein gel can reveal the degree of interaction and aggregation of protein molecules, and can also reflect the degree of protein denaturation. The results showed that the microstructures of experimental groups 2 and 3 were tight and smooth, while the structure of experimental group 1 was the loosest and roughest. This shows that oxidation can destroy the network structure of protein gel. Experimental groups 2 and 3 can inhibit protein oxidation, thereby inhibiting structural damage. After compounding, the network structure of the gel will be more uniform, the porosity will be reduced, and the texture of the gel will be improved. Compared with experimental group 3, experimental group 2 can form more connection points with protein molecules, enhancing the continuity and integrity of the gel network, indicating that the polypeptide NPAAKMSPKPATP can promote protein cross-linking and reduce the formation of large pores on the one hand, and fill small pores on the other hand, making the gel structure more uniform and dense.

[0059] The above description is only a preferred specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any technician familiar with the technical field, within the technical scope disclosed by the present invention, who makes equivalent replacements or changes based on the technical solution and inventive concept of the present invention, should be covered by the scope of protection of the present invention.

Claims

1. A polypeptide NPAAKMSPKPATP, characterized in that: The steps include: S1. Preparation of Paenibacillus Provence PP-2 metabolites: After solidification, Paenibacillus Provence PP-2 was inoculated and streaked on LB solid medium, then incubated upside down in a constant temperature incubator for 48 hours. A single colony was picked and inoculated into LB liquid medium, incubated on a sterile shaker for 24 hours, and then inoculated into LB liquid medium at a 1% addition rate and incubated for 7 days. The culture was then centrifuged at 8000 rpm and 4°C for 10 minutes, the precipitate was discarded, and the supernatant was sterilized by filtration using a 0.22 μm filter membrane to obtain Paenibacillus Provence PP-2 metabolites. S2. Isolating and purifying the polypeptide NPAAKMSPKPATP from the metabolites in step S1: Using gel column chromatography to further screen the antibacterial components of the Paenibacillus Provence PP-2 metabolites to obtain peptide fragments, and then identifying and analyzing the peptide sequences. After analysis, the polypeptide NPAAKMSPKPATP is synthesized.

2. A polypeptide NPAAKMSPKPATP according to claim 1, characterized in that: In step S2, the peptide sequence is identified by LC-MS method, and the mobile phase A of the peptide sequence is 0.1% formic acid and 2% acetonitrile; the mobile phase B is 0.1% formic acid and 80% acetonitrile.

3. A polypeptide NPAAKMSPKPATP according to claim 1, characterized in that: In step S2, the peptide sequence is analyzed de novo using a Q-Exactive HF-X mass spectrometer in a data-dependent acquisition mode for mass spectrometry analysis.

4. A polypeptide NPAAKMSPKPATP according to claim 1, characterized in that: In step S2, the polypeptide NPAAKMSPKPATP is synthesized using the Fmoc solid phase synthesis method.

5. Use of the polypeptide NPAAKMSPKPATP according to any one of claims 1 to 4 in a biological preservative.

6. Use of the polypeptide NPAAKMSPKPATP in a biological preservative according to claim 5, characterized in that: The preparation method of the biological preservative containing the polypeptide NPAAKMSPKPATP is as follows: the polypeptide NPAAKMSPKPATP powder is dissolved in sterile distilled water, and proanthocyanidins and agar oligosaccharides are added and mixed evenly to obtain a composite biological preservative.

7. Use of the polypeptide NPAAKMSPKPATP in a biological preservative according to claim 6, characterized in that: The concentration of the polypeptide NPAAKMSPKPATP powder after being dissolved in sterile distilled water is 180 mg / mL.

8. The use of the polypeptide NPAAKMSPKPATP in a biological preservative according to claim 6, characterized in that: The mass concentration of the proanthocyanidins is 0.2% w / v.

9. Use of the polypeptide NPAAKMSPKPATP in a biological preservative according to claim 6, characterized in that: The mass concentration of the agar oligosaccharide is 0.5% w / v.

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