An oligopeptide and its application in modifying lysozyme to enhance antibacterial spectrum and antibacterial activity
By coupling oligopeptides with lysozyme, the antibacterial activity of lysozyme against Gram-positive and Gram-negative bacteria is enhanced, solving the problem of poor efficacy of existing lysozymes against Gram-negative bacteria, and achieving broad-spectrum antibacterial effect and safe food preservation application.
Patent Information
- Application Number
- CN202510015868.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2025-01-06
- Publication Date
- 2025-11-21
- Estimated Expiration
- 2045-01-06
AI Technical Summary
Existing lysozymes have poor inhibitory effects on Gram-negative bacteria, and existing modification methods have issues such as strict operating conditions or the introduction of chemical reagents leading to low safety, which limits their industrialization and promotion in the food and pharmaceutical fields.
An oligopeptide is coupled with lysozyme to enhance the antibacterial activity of lysozyme against Gram-positive and Gram-negative bacteria. The high affinity binding between the oligopeptide and lysozyme is verified by biomembrane interference molecular interaction method. Modified lysozyme is prepared for use in the preparation of antibacterial and preservative products.
Oligopeptide-modified lysozyme has a significant inhibitory effect on both Gram-positive and Gram-negative bacteria, expanding the antibacterial spectrum of lysozyme and improving its antibacterial and preservative effects in the food and biopharmaceutical fields.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology and relates to an oligopeptide that can bind to lysozyme and enhance lysozyme activity and antibacterial spectrum, as well as its applications. Background Technology
[0002] Lysozyme, also known as cell wall enzyme, is widely found in nature and possesses antiviral, antibacterial, analgesic, anti-swelling, and tissue repair-accelerating functions. Due to its biocompatibility and non-irritating, non-toxic properties, it has been formulated into a high-quality enzyme preparation that can kill pathogenic microorganisms without damaging the organism, and is widely used in medicine, food, feed, daily chemicals, and biological research. In the field of food preservation, lysozyme is considered a safe food additive. Adding lysozyme can kill bacteria and prevent spoilage. Because it does not require heating, it avoids the destructive effects of high-temperature sterilization on food flavor, making it particularly important for the preservation of heat-sensitive foods.
[0003] Egg white lysozyme is a single peptide chain composed of 129 amino acids, cross-linked with four pairs of disulfide bonds. It can disrupt the peptidoglycan structure of bacteria, causing cell wall rupture, leakage of contents, and bacterial lysis, thereby achieving sterilization. However, due to Gram-negative bacteria (G... － The cell wall of *Glycine somniferum* contains an LPS layer, which makes it resistant to natural lysozyme. Therefore, natural lysozyme is only resistant to *Glycine somniferum*. + The bacteria have a significant bacteriolytic effect, while they have a significant effect on G. － Lysozyme has almost no antibacterial effect or its effect is not very good. To broaden the application of lysozyme in food antibacterial treatment, scholars at home and abroad have been continuously trying various modifications and alterations to lysozyme over the past 20 years to improve its antibacterial efficacy against Gram-negative bacteria. The main modification methods include: First, partial thermal denaturation of lysozyme, using reducing agents to reduce the disulfide bonds of lysozyme, or hydrolyzing lysozyme into small polypeptide molecules; Second, combining lysozyme with physical methods such as high-pressure or ultra-high-pressure homogenization of cells to change cell morphology, reduce outer membrane resistance, and facilitate lysozyme entry into the peptidoglycan layer; Third, adding penetrants or reducing agents such as EDTA and Na2SO4 to synergistically enhance the antibacterial activity of lysozyme to varying degrees. However, these methods generally suffer from strict operating conditions or low safety due to the introduction of chemical reagents, making them unsuitable for industrial-scale promotion in the food and pharmaceutical fields. Therefore, there is an urgent need to propose more targeted strategies to improve the preservative and freshness-preserving effects of lysozyme. Summary of the Invention
[0004] The purpose of this invention is to overcome the above-mentioned defects and provide an oligopeptide and its application in enhancing the antibacterial spectrum and antibacterial activity of modified lysozyme.
[0005] This invention provides an egg-derived oligopeptide that can enhance lysozyme activity. Experimental verification has shown that oligopeptide I, oligopeptide II, oligopeptide III, and oligopeptide IV provided by this invention can be coupled with egg white lysozyme, thereby improving the antibacterial activity of natural lysozyme. The coupled lysozyme has an inhibitory effect on both Gram-positive and Gram-negative bacteria and can be used to prepare antibacterial and preservative products, solving the problem of antibacterial preservation of heat-sensitive foods.
[0006] The oligopeptide provided by this invention is oligopeptide I and / or oligopeptide II;
[0007] The amino acid sequence of the oligopeptide I is: SASPTSPPR;
[0008] The amino acid sequence of the oligopeptide II is: PSPVLSTPEFRVP.
[0009] This invention demonstrates, through the biomembrane interference molecular interaction method, that the oligopeptide provided by this invention can bind to lysozyme with extremely high affinity. A lysozyme activity assay kit was used to find that it enhances the antibacterial spectrum and activity of lysozyme. This invention can be applied in the fields of biomedicine and food preservation to enhance the antibacterial spectrum and activity of lysozyme.
[0010] The modified lysozyme obtained by coupling the oligopeptide provided by this invention with natural lysozyme has a higher conformation than natural lysozyme. Antibacterial effect experiments of this invention have shown that the oligopeptide-modified lysozyme has significant inhibitory effects on Gram-negative bacteria such as *Escherichia coli* and *Salmonella*, as well as *Staphylococcus aureus* and *Listeria monocytogenes*. Compared with natural lysozyme (natural egg white lysozyme), the oligopeptide-modified lysozyme has a broader antibacterial spectrum and enhanced antibacterial activity, and can be used to prepare antibacterial and / or preservative products for treating bacterial infections, preventing food spoilage, and improving the antibacterial properties of biopharmaceutical and food preservative products. Attached Figure Description
[0011] Figure 1 This is a BIL analysis diagram of the interaction between four oligopeptides and egg white lysozyme. In the diagram, A represents the BIL analysis of the interaction between oligopeptide I and egg white lysozyme, B represents the BIL analysis of the interaction between oligopeptide II and egg white lysozyme, C represents the BIL analysis of the interaction between oligopeptide III and egg white lysozyme, and D represents the BIL analysis of the interaction between oligopeptide IV and egg white lysozyme. Detailed Implementation
[0012] Specific implementation method one: This implementation method is an oligopeptide, specifically oligopeptide I and / or oligopeptide II;
[0013] The amino acid sequence of the oligopeptide I is: SASPTSPPR;
[0014] The amino acid sequence of the oligopeptide II is: PSPVLSTPEFRVP.
[0015] Specific Implementation Method Two: This implementation method differs from Specific Implementation Method One in that the oligopeptide is obtained by separating and purifying the egg liquid extract fermented by Lactobacillus plantarum Lp05. The other steps are the same as in Specific Implementation Method One.
[0016] Specific Implementation Method Three: This implementation method differs from Specific Implementation Method One or Two in that the oligopeptide is synthesized using a solid-phase synthesis method based on its amino acid sequence or obtained by expressing its encoding gene in engineered bacterial strains. The other steps are the same as in Specific Implementation Method One or Two.
[0017] Specific Implementation Method Four: This implementation method differs from Specific Implementation Methods One to Three in that the oligopeptide is used in enhancing the antibacterial spectrum and antibacterial activity of the modified lysozyme. The other steps are the same as in Specific Implementation Methods One to Three.
[0018] Specific Implementation Method Five: This implementation method differs from Specific Implementation Methods One to Four in that: the oligopeptide is coupled or mixed with natural lysozyme to improve the antibacterial spectrum and antibacterial activity of the natural lysozyme. The other steps are the same as in Specific Implementation Methods One to Four.
[0019] Specific Implementation Method Six: This implementation method differs from Specific Implementation Methods One to Five in that: the oligopeptide is coupled or mixed with natural lysozyme and then used in the preparation of broad-spectrum antibacterial and / or preservative products. The other steps are the same as in Specific Implementation Methods One to Five.
[0020] Specific Implementation Method Seven: This implementation method differs from Specific Implementation Methods One to Six in that the product described is a food additive, feed additive, and / or antibacterial drug. The other steps are the same as in Specific Implementation Methods One to Six.
[0021] The beneficial effects of the present invention are verified using the following embodiments:
[0022] Example 1: The process of obtaining protein-derived oligopeptides (Oligopeptide I, Oligopeptide II, Oligopeptide III, Oligopeptide IV):
[0023] Fresh eggs were shelled and then freeze-dried. 20g of the resulting freeze-dried egg powder was weighed and added to 350mL of deionized water, stirred thoroughly, and then incubated at 110℃ for 30min, followed by cooling to 30℃. The reaction solution was inoculated in a clean bench with an initial colony count of approximately 10⁻⁶. 8The mixture of *Lactobacillus plantarum* culture medium (purchased from Wecon Probiotics Co., Ltd.) at CFU / mL was fermented at 37°C for 14 h with constant shaking. After fermentation, the reaction was terminated by heating at 95°C for 15 min and then immediately cooling. The reaction mixture was centrifuged at 10,000 × g for 15 min, and the supernatant was collected. The supernatant was desalted and analyzed on Ultra-High Resolution LC-MS. The LC-MS conditions were: Phase A: 0.1% formic acid; Phase B: 0.1% formic acid, 80% acetonitrile; flow rate: 300 nL / min. The mass spectrometry conditions were: positive ion mode, primary mass spectrometry resolution: 12000; scan range: 350-1800; secondary mass spectrometry resolution: 3000, scan mode: auto; HCD collision energy (%): 30. The raw mass spectrometry file was retrieved from the target protein database using Proteome Discovered 2.2: gallusNCBI20240514. A total of 1024 peptide amino acid sequences were obtained. The retrieved peptides were analyzed using the online tool PeptideRanker (http: / / distilldeep.ucd.ie / PeptideRanker / ) for solubility, activity, and toxicity prediction. 135 bioactive peptides with good solubility and activity scores higher than 0.5 were identified. These bioactive peptides were then subjected to batch docking with Autodock Vina, identifying amino acid sequences SEQ ID 1–4 (SEQ ID NO.1: SASPTSPPR; SEQ ID NO.2: PSPVLSTPEFRVP; SEQ ID NO.3: TPPLTGDFR; SEQ ID NO.4: DWLSGERF) with potential affinity for lysozyme. The amino acid sequences of these peptides were then entered into the BIOPEP database (https: / / biochemia.uwm.edu.pl / biopep-uwm / ) to search for matching known functional peptides. No matching homologous peptides were found, indicating that these are novel oligopeptides. Based on the amino acid sequences shown, active peptides (oligopeptide I, oligopeptide II, oligopeptide III, and oligopeptide IV) with amino acid sequences such as SEQ ID 1–4 were synthesized using conventional solid-phase synthesis methods, with the purity of the synthesized peptides >90% (the proportion of the target peptide in the total peptides).
[0024] The amino acid sequence of oligopeptide I (SEQ ID 1) is: SASPPTPR;
[0025] The amino acid sequence (SEQ ID2) of oligopeptide II is: PSPVLSTPEFRVP;
[0026] The amino acid sequence (SEQ ID3) of oligopeptide III is: TPPLTGDFR;
[0027] The amino acid sequence of oligopeptide IV is (SEQ ID 4): DWLSGERF.
[0028] Example 2: Experimental verification of the coupling effect between protein-derived oligopeptides and lysozyme:
[0029] The interaction between four oligopeptides (oligopeptide I, oligopeptide II, oligopeptide III, and oligopeptide IV) and egg white lysozyme was analyzed using a biomembrane interference molecular interaction analyzer (Molecular Devices, LLC, Octet RED96e). Non-specific binding was excluded using bovine serum albumin (BSA). The experiment was conducted at 30°C with a shaker speed of 1000 rpm. After 20 min of pre-hydration and 1 min of sensor inspection of the AR2G biosensor, the sensor was activated by immersing it in a mixed solution containing 20 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride and 10 mM N-hydroxysuccinimide for 5 min. Lysozyme in sodium acetate buffer (pH 6.0) was immobilized on the activated AR2G biosensor, followed by blocking with 1 M ethanolamine (pH 8.5) for 5 min. After baseline equilibration for 2 min, binding was performed with 50-200 nM amino acid sequences such as SEQ ID 1-4 oligopeptides (oligopeptide I, oligopeptide II, oligopeptide III, and oligopeptide IV) for 5 min, followed by separation in the same buffer used for baseline equilibration for 5 min. Data were fitted 1:1 using Ddata Analysis software, and the results are as follows: Figure 1 As shown.
[0030] Figure 1 The diagram shows the BIL analysis of the interaction between four oligopeptides and egg white lysozyme. In the diagram, A represents the BIL analysis of the interaction between oligopeptide I and egg white lysozyme, B represents the BIL analysis of the interaction between oligopeptide II and egg white lysozyme, C represents the BIL analysis of the interaction between oligopeptide III and egg white lysozyme, and D represents the BIL analysis of the interaction between oligopeptide IV and egg white lysozyme.
[0031] Figure 1 The increase in the mid-curve signal indicates that the oligopeptide's ability to bind to lysozyme immobilized on the sensor leads to a higher signal. The decrease in the later part of the curve is due to the separation of unbound molecules from the lysozyme during buffer washing. The results showed that all four peptides used (oligopeptide I, oligopeptide II, oligopeptide III, and oligopeptide IV) could bind to lysozyme nonspecifically with high affinity, which may cause conformational changes in the lysozyme.
[0032] Example 3: Preparation process of oligopeptide-coupled natural lysozyme:
[0033] Egg white lysozyme (purchased from Xinjiang Xipa Health Food Co., Ltd.) was weighed and dissolved in glycine buffer (50 mmol / L) at pH 5.0 to prepare a concentration of 0.5 mg / mL. The oligopeptides with sequences as shown in SEQ ID 1–4 obtained in Example 1 were then added, and the mixture was incubated at 37°C with shaking for 12 h at a shaking speed of 250 r / min. After incubation, the free oligopeptides were removed using an ultrafiltration tube with a molecular weight cutoff of 3 kDa. The retentate containing lysozyme was freeze-dried to obtain oligopeptide-coupled natural lysozyme (oligopeptide-coupled modified lysozyme).
[0034] Example 4: Evaluation of the effect of oligopeptide-modified natural lysozyme on lysing activity:
[0035] The enhancing effect of synthesized oligopeptides on lysozyme activity was detected according to the instructions of the lysozyme activity assay kit. Specifically, 200 μL of natural lysozyme (enzyme activity 20000 U / mg) at concentrations of 0.05 and 0.5 mg / mL, the oligopeptide obtained in Example 1, and the oligopeptide-coupled natural lysozyme obtained in Example 3 (oligopeptide-coupled modified lysozyme) were added to 96-well plates respectively. After incubation at 37°C for 1 h, the plates were cooled for 5 min, and then a pre-adjusted *Micrococcus lysinensis* bacterial suspension (the absorbance of the *Micrococcus lysinensis* suspension was adjusted to 1.0 according to the method specified in GB / T 30990) was added. Incubation was continued at 37°C for 15 min, and the transmittance at 530 nm was measured. Higher sample transmittance indicates stronger lysolytic activity of the lysozyme.
[0036] Table 1 Comparison of lysozyme activity between natural lysozyme and modified lysozyme
[0037]
[0038] As shown in the table above, the transmittance of the bacterial culture was very low after adding the synthetic oligopeptides alone, indicating that the individual peptides did not have significant lysing activity. Compared with natural egg white lysozyme, oligopeptide I-conjugated modified lysozyme L1 and oligopeptide II-conjugated modified lysozyme L2 had higher transmittance, indicating that they could effectively increase the lysing activity of the lysozyme. However, oligopeptide III-conjugated modified lysozyme L3 and oligopeptide IV-conjugated modified lysozyme L4 did not show a significant enhancement in lysing activity.
[0039] Example 5: Antibacterial activity of protein-derived oligopeptide-modified lysozyme against different bacteria:
[0040] The oligopeptide-conjugated modified lysozyme prepared in Example 3 was collected. A series of gradient solutions of lysozyme, oligopeptides (Oligopeptide I and Oligopeptide II) with sequences SEQ ID 1–2, and oligopeptide-conjugated modified lysozyme (solvent: physiological saline) were prepared using physiological saline as the diluent (5, 2.5, 1.25, 0.625, 0.313, 0.157, 0.078, 0.039, 0.0196, 0.01 mg / mL). Indicator bacteria such as Staphylococcus aureus, Escherichia coli, and Listeria monocytogenes were cultured overnight and diluted with LB medium to a final bacterial density of 1.0 × 10⁻⁶. 8 Approximately CFU / mL. 10 μL of each diluted concentration of lysozyme and modified lysozyme was added to a 96-well plate containing 90 μL of bacterial solution. A growth control and a sterile control were set up. The plates were incubated at 37℃ for 20 h. The minimum inhibitory concentration was determined based on the turbidity of the bacterial solution in the wells. The results are shown in Table 2.
[0041] Table 2. Determination of the minimum inhibitory concentration of modified lysozyme against various foodborne pathogens.
[0042]
[0043]
[0044] It was found that the oligopeptides themselves had weak antibacterial activity, with a minimum inhibitory concentration (MIC) greater than 5 mg / mL against the tested strains. The MIC of lysozyme modified with oligopeptide I and oligopeptide II was significantly reduced. The oligopeptide-coupled modified lysozyme not only had a lower MIC against Gram-positive bacteria such as Staphylococcus aureus and Micrococcus luteus, but also had an inhibitory effect on Gram-negative bacteria such as Salmonella. This indicates that the coupling modification of SEQ ID 1-2 oligopeptides (oligopeptide I and oligopeptide II) significantly improved the antibacterial spectrum and antibacterial activity of lysozyme.
[0045] Example 6: Antibacterial effect of oligopeptide-conjugated modified lysozyme on bacteria on the surface of fresh beef
[0046] Natural lysozyme and oligopeptide-conjugated modified lysozyme L1 and L2 at concentrations of 0.05–5 mg / mL were sprayed onto the surface of beef. The beef was then covered with plastic wrap and placed in a 4°C refrigerator and a 25°C incubator, respectively. After 2 hours, the number of colonies in the samples was determined using the conventional colony counting method. The results showed that both lysozyme and oligopeptide-conjugated modified lysozyme L1 reduced the total number of spoilage bacteria colonies on the surface of beef. At 25°C, compared with using 5 mg / mL natural lysozyme alone, the lysozyme modified with oligopeptides (oligopeptide I and oligopeptide II) of SEQ ID 1 and 2 reduced the spoilage bacteria biofilm on the surface of beef by 2–4 log CFU / g (Table 3).
[0047] Table 3. Number of bacterial biofilms on beef 2 hours after treatment with lysozyme and modified lysozyme.
[0048]
[0049]
Claims
1. An oligopeptide, characterized in that... The amino acid sequence of the oligopeptide is: SASPTSPPR.
2. The method for preparing an oligopeptide according to claim 1, characterized in that... The oligopeptides are synthesized using solid-phase synthesis based on their amino acid sequences or obtained by expressing their encoding genes in engineered bacterial strains.
3. The application of the oligopeptide as described in claim 1, characterized in that... The oligopeptide is mixed with egg white lysozyme and incubated before being used in the preparation of broad-spectrum antibacterial and / or preservative products, which are targeted at Gram-positive or Gram-negative bacteria.
4. The application of the oligopeptide according to claim 3, characterized in that... The products mentioned are food additives, feed additives, and / or antibacterial drugs.