Oligopeptide and application thereof in enhancement of antibacterial spectrum and antibacterial activity of modified lysozyme

Abstract

The invention discloses oligopeptide and application thereof in enhancing antibacterial spectrum and antibacterial activity by modifying lysozyme, and belongs to the technical field of biology. The oligopeptide is oligopeptide I and / or oligopeptide II, and amino acid sequences of the oligopeptide I and / or oligopeptide II are shown as SEQ ID NO.1-2. The oligopeptide for enhancing the antibacterial activity of the lysozyme is screened and identified from the lactobacillus plantarum Lp05 fermented egg liquid for the first time. After a peptide sequence which is non-toxic, high in water solubility and capable of enhancing the activity of the lysozyme is obtained through LC-MS / MS identification, ToxinPred, admetSAR and BIOPEP-UWM program analysis and molecular docking screening, the peptide sequence is synthesized, and experimental verification for improving the activity of the lysozyme and an antibacterial spectrum is carried out. The oligopeptide and the lysozyme are coupled, the prepared oligopeptide coupling modified lysozyme achieves the effect of remarkably improving the antibacterial spectrum and the antibacterial activity of the lysozyme, and the oligopeptide coupling modified lysozyme can be applied to enhancing the antibacterial spectrum and the antibacterial activity of the lysozyme in the fields of biological medicine and food preservation.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to an oligopeptide capable of combining with lysozyme and improving the activity and antibacterial spectrum of lysozyme and an application thereof. Background Art

[0002] Lysozyme, also known as cell wall enzyme, is widely present in nature and has antiviral, antibacterial, analgesic, detumescent, and tissue repair-accelerating functions. Due to its biocompatibility and non-irritating and non-toxic characteristics, it has been made into an enzyme preparation with excellent properties that can kill pathogenic microorganisms without damaging the body. It is widely used in medical treatment, food, feed, daily chemicals, and biological research. In the field of food preservation, lysozyme is considered to be a safe food additive. Adding lysozyme can kill bacteria and preserve food. Since it does not require heating, it avoids the destructive effect of high-temperature sterilization on food flavor. It is especially important for the preservation of heat-sensitive foods.

[0003] Egg white lysozyme is a single peptide chain composed of 129 amino acids, with 4 pairs of disulfide cross-links. It can destroy the peptidoglycan structure of bacteria, causing cell wall rupture, leakage of contents and dissolution of bacteria, thereby achieving sterilization. － ) has an LPS layer outside the cell wall, which is resistant to natural lysozyme. Therefore, natural lysozyme is only resistant to G + It has a significant bacteriolytic effect on G － There is almost no antibacterial effect or the effect is not very good. In order to broaden the application scope of lysozyme in food antibacterial, domestic and foreign scholars have been trying to modify and modify lysozyme in various ways in the past 20 years to improve its antibacterial effect on G-. The main modification methods are: first, partial heat denaturation of lysozyme, using reducing agents to reduce the disulfide bonds of lysozyme, or hydrolyzing lysozyme into small molecular polypeptides; second, while using lysozyme to inhibit bacteria, physical methods such as high pressure and ultra-high pressure homogenization to treat cells are combined to change the cell morphology, reduce the outer membrane resistance, and facilitate lysozyme to enter the peptidoglycan layer; third, adding EDTA, Na2SO4 and other permeabilizing agents or reducing agents to lysozyme synergistically will also improve the antibacterial activity of lysozyme to varying degrees. However, these methods generally have problems such as strict operating conditions or low safety caused by the introduction of chemical reagents, and are not suitable for industrial promotion in the fields of food and medicine. Therefore, it is urgent to propose more targeted strategies to improve the antiseptic and fresh-keeping effect of lysozyme. Summary of the invention

[0004] The purpose of the present 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] The present invention provides an egg-derived oligopeptide capable of enhancing the activity of lysozyme. Experimental verification shows that the oligopeptides I, II, III and IV provided by the present 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 antiseptic products, thereby solving the problem of antibacterial and fresh-keeping of heat-sensitive foods.

[0006] An oligopeptide provided by the present 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] The present invention proves that the oligopeptide provided by the present invention can bind to lysozyme with extremely high affinity through the biomembrane interference molecular interaction method, and finds through the lysozyme activity detection kit that the antibacterial spectrum and antibacterial activity of lysozyme are improved. It can be applied in the fields of biomedicine and food preservation to enhance the antibacterial spectrum and antibacterial activity of lysozyme.

[0010] The modified lysozyme obtained by coupling the oligopeptide provided by the present invention with natural lysozyme has a higher conformation different from that of natural lysozyme. The present invention has found through antibacterial effect experiments that the oligopeptide-modified lysozyme has a significant inhibitory effect 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 wide antibacterial spectrum and enhanced antibacterial activity, and can be used to prepare antibacterial and / or antiseptic products for treating bacterial infections, preventing food corruption, and improving the antibacterial properties of biopharmaceuticals and food antiseptic products. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Figure 1 is a BIL analysis diagram of the interaction between four oligopeptides and egg white lysozyme. Figure A is the BIL analysis of the interaction between oligopeptide I and egg white lysozyme, B is the BIL analysis of the interaction between oligopeptide II and egg white lysozyme, C is the BIL analysis of the interaction between oligopeptide III and egg white lysozyme, and D is the BIL analysis of the interaction between oligopeptide IV and egg white lysozyme. DETAILED DESCRIPTION

[0012] Specific embodiment 1: This embodiment is an oligopeptide, which is 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 embodiment 2: This embodiment differs from specific embodiment 1 in that the oligopeptide is obtained by separating and purifying the egg extract after fermentation with Lactobacillus plantarum Lp05. The other steps are the same as those of specific embodiment 1.

[0016] Specific embodiment 3: This embodiment differs from specific embodiment 1 or 2 in that the oligopeptide is synthesized by solid phase synthesis according to the amino acid sequence or obtained by expressing its encoding gene in an engineered bacterial strain. The other steps are the same as those of specific embodiment 1 or 2.

[0017] Specific embodiment 4: This embodiment differs from specific embodiments 1 to 3 in that the oligopeptide is used in the modification of lysozyme to enhance the antibacterial spectrum and antibacterial activity. The other steps are the same as those of specific embodiments 1 to 3.

[0018] Specific embodiment 5: This embodiment differs from specific embodiments 1 to 4 in that the oligopeptide is coupled or mixed with natural lysozyme to improve the antibacterial spectrum and antibacterial activity of natural lysozyme. The other steps are the same as those of specific embodiments 1 to 4.

[0019] Specific embodiment 6: This embodiment differs from specific embodiments 1 to 5 in that the oligopeptide is coupled or mixed with natural lysozyme and then used in the preparation of a broad-spectrum antibacterial and / or antiseptic product. The other steps are the same as those of specific embodiments 1 to 5.

[0020] Specific embodiment 7: This embodiment differs from specific embodiments 1 to 6 in that the product is a food additive, a feed additive and / or an antibacterial drug. The other steps are the same as those of specific embodiments 1 to 6.

[0021] The following examples are used to verify the beneficial effects of the present invention:

[0022] Example 1: Process for obtaining egg-derived oligopeptides (oligopeptide I, oligopeptide II, oligopeptide III, oligopeptide IV):

[0023] Fresh eggs were shelled and freeze-dried. 20 g of freeze-dried egg powder was weighed and added to 350 mL of deionized water. The mixture was stirred evenly and then kept at 110°C for 30 min and cooled to 30°C. The reaction solution was inoculated in a clean bench with an initial colony count of about 10 8CFU / mL of plant lactobacillus (purchased from Weikang Probiotics Co., Ltd.) culture fluid, and the mixture was fermented and cultured at 37°C for 14h while shaking continuously. After the fermentation was completed, the fermentation reaction was terminated by heating at 95°C for 15min and immediately cooling. The reaction mixture was centrifuged at 10,000×g for 15min, and the supernatant was collected. The supernatant was desalted and analyzed on Ultra-High Resolution LC-MS, where the liquid chromatography conditions were: phase A: 0.1% formic acid; phase B: 0.1% formic acid, 80% acetonitrile; flow rate: 300nL / min. Mass spectrometry conditions: positive ion mode, primary mass spectrometry resolution: 12000; scanning range: 350-1800; secondary mass spectrometry resolution: 3000, scanning mode automatic, HCD collision energy (%): 30. The mass spectrometry raw file used Proteome Discovered2.2 to retrieve the target protein database: gallusNCBI20240514. Results A total of 1024 amino acid sequences of peptides were obtained. The retrieved peptides were used to predict the solubility, activity and toxicity using the online tool PeptideRanker (http: / / distilldeep.ucd.ie / PeptideRanker / ). A total of 135 active peptides with good solubility and activity scores higher than 0.5 were screened. These active peptides were batch docked using Autodock Vina, and 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 were identified. The amino acid sequences of these peptides were 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 they are novel oligopeptides. According to the amino acid sequence shown, active peptides (oligopeptide I, oligopeptide II, oligopeptide III, oligopeptide IV) with amino acid sequences such as SEQ ID 1 to 4 were synthesized by conventional solid phase synthesis methods, and the purity of the synthetic peptides was >90% (the proportion of the target peptide in the total peptides).

[0024] The amino acid sequence of oligopeptide I (SEQ ID 1) is: SASPTSPPR;

[0025] The amino acid sequence of oligopeptide II (SEQ ID2) is: PSPVLSTPEFRVP;

[0026] The amino acid sequence of oligopeptide III (SEQ ID 3) 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 egg-derived oligopeptides and lysozyme:

[0029] The interaction between four oligopeptides (oligopeptide I, oligopeptide II, oligopeptide III, oligopeptide IV) and egg white lysozyme was analyzed using a biomembrane interferometer (Octet RED96e, Molecular Devices, LLC, USA). Bovine serum albumin (BSA) was used to exclude nonspecific binding. The test was carried out at 30°C with a shaker speed of 1000 rpm. After the AR2G biosensor was prehydrated for 20 minutes and the sensor was checked for 1 minute, the sensor was immersed in a mixed solution containing 20mM 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride and 10mM N-hydroxysuccinimide for 5 minutes of activation. 20μg / mL lysozyme in sodium acetate buffer (pH 6.0) was immobilized on the activated AR2G biosensor, and then blocked with 1M ethanolamine (PH 8.5) for 5 minutes. After 2 minutes of baseline equilibration, it was combined with 50-200nM amino acid sequences such as SEQ ID 1 to 4 oligopeptides (oligopeptide I, oligopeptide II, oligopeptide III, oligopeptide IV) for 5 minutes, and then separated in the same buffer used for baseline equilibration for 5 minutes. Ddata Analysis software was used for 1:1 data fitting processing, and the results are shown in Figure 2. Figure 1 shown.

[0030] Figure 1 Figure 1 is a BIL analysis diagram of the interaction between four oligopeptides and egg white lysozyme, in which A is the BIL analysis of the interaction between oligopeptide I and egg white lysozyme, B is the BIL analysis of the interaction between oligopeptide II and egg white lysozyme, C is the BIL analysis of the interaction between oligopeptide III and egg white lysozyme, and D is the BIL analysis of the interaction between oligopeptide IV and egg white lysozyme;

[0031] Figure 1 The increase in the middle curve signal indicates that if the oligopeptide can bind to the lysozyme fixed on the sensor, it will lead to an increase in the curve signal. The decrease in the signal at the end of the curve is because the unbound molecules are separated from the lysozyme during the buffer wash. The results showed that the four peptides used (oligopeptide I, oligopeptide II, oligopeptide III, and oligopeptide IV) can all bind to lysozyme nonspecifically with high affinity, and this binding may cause a conformational change in 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 a pH 5.0 glycine buffer (50mmol / L) to a mass concentration of 0.5mg / mL, and the oligopeptides obtained in Example 1 as shown in SEQ ID1-4 were added respectively, and incubated at 37°C for 12h with an oscillation speed of 250r / min. After the incubation, the free oligopeptides were removed with an ultrafiltration tube with a molecular weight cutoff of 3kDa, and 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 modification on the lysozyme activity of natural lysozyme:

[0035] According to the operating instructions of the lysozyme activity assay kit, the synthetic oligopeptide was tested for its enhancing effect on lysozyme activity. The specific method is as follows: 200 μL of natural lysozyme (enzyme activity of 20000 U / mg) with concentrations of 0.05 and 0.5 mg / mL, the oligopeptide obtained by Example 1, and the oligopeptide-coupled natural lysozyme (oligopeptide-coupled modified lysozyme) obtained by Example 3 were added to a 96-well plate, incubated at 37 ° C for 1 h and then cooled to 5 min, and then the lysodeiktic micrococcal suspension with pre-adjusted bacterial liquid concentration was added (according to the method specified in GBT30990, the absorbance of the lysodeiktic micrococcal suspension was adjusted to 1.0), continued to incubate at 37 ° C for 15 min, and measured the transmittance at 530 nm. The higher the sample transmittance, the stronger the lysozyme activity.

[0036] Table 1 Comparison of lysozyme activity between natural lysozyme and modified lysozyme

[0037]

[0038] As can be seen from the table above, after adding the synthetic oligopeptide alone, the transmittance of the bacterial solution is very low, indicating that the single peptide has no obvious lysozyme activity. Compared with natural egg white lysozyme, oligopeptide I coupled modified lysozyme L1 and oligopeptide II coupled modified lysozyme L2 have higher transmittance, indicating that they can effectively increase the lysozyme lysozyme activity. However, the lysozyme activity of oligopeptide III coupled modified lysozyme L3 and oligopeptide IV coupled modified lysozyme L4 was not significantly enhanced.

[0039] Example 5: Antibacterial activity of egg-derived oligopeptide-modified lysozyme against different bacteria:

[0040] The oligopeptide-coupled modified lysozyme prepared in Example 3 was collected. Using physiological saline as the diluent, a series of gradient lysozymes, oligopeptides (oligopeptide I, oligopeptide II) with sequences of SEQ ID 1-2, and oligopeptide-coupled modified lysozyme solutions (solvent is physiological saline) were prepared by double dilution method (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 8 CFU / mL. Take 10 μL of diluted lysozyme and modified lysozyme of each concentration and add them to a 96-well plate containing 90 μL of bacterial solution, set up a growth control and a sterile control, and culture at 37°C for 20 hours. The minimum inhibitory concentration is determined according to the turbidity of the bacterial solution in the well. The results are shown in Table 2.

[0041] Table 2 Determination of minimum inhibitory concentration of modified lysozyme against various foodborne pathogens

[0042]

[0043]

[0044] It can be found that the oligopeptide itself has weak antibacterial activity, and the minimum inhibitory concentration for the test strain is greater than 5 mg / mL. The minimum inhibitory concentration of lysozyme modified by oligopeptide I and oligopeptide II is significantly reduced. The oligopeptide-coupled modified lysozyme not only has a lower inhibitory concentration for Gram-positive bacteria such as Staphylococcus aureus and Micrococcus luteus, but also has an inhibitory effect on Gram-negative bacteria such as Salmonella. It also shows that the coupling modification of SEQ ID 1-2 oligopeptides (oligopeptide I, oligopeptide II) significantly improves the antibacterial spectrum and antibacterial activity of lysozyme.

[0045] Example 6: Antibacterial effect of oligopeptide-coupled modified lysozyme on bacteria on the surface of fresh beef

[0046] The natural lysozyme and oligopeptide-coupled modified lysozyme L1 and L2 at a concentration of 0.05-5 mg / mL were sprayed on the surface of beef, and then the beef was covered with plastic wrap and placed in a 4°C refrigerator and a 25°C incubator, respectively. After 2 hours, the beef was taken out and the number of colonies in the samples was determined by the conventional colony counting method. The results showed that both lysozyme and oligopeptide I-coupled modified lysozyme L1 could reduce the total number of spoilage bacteria on the surface of beef. At 25°C, compared with the use of 5 mg / mL natural lysozyme alone, the lysozyme coupled with oligopeptides (oligopeptide I and oligopeptide II) with sequences of SEQ ID 1 and 2 could reduce the biofilm of spoilage bacteria on the surface of beef by 2-4 log CFU / g, respectively (Table 3).

[0047] Table 3 The number of bacterial biofilms on beef after lysozyme and modified lysozyme treatment for 2 hours

[0048]

[0049]

Claims

1. An oligopeptide, characterized in that The oligopeptide is oligopeptide I and / or oligopeptide II; The amino acid sequence of the oligopeptide I is: SASPTSPPR; The amino acid sequence of the oligopeptide II is: PSPVLSTPEFRVP.

2. An oligopeptide according to claim 1, characterized in that The oligopeptide is obtained by separating and purifying an egg liquid extract fermented with plant lactobacillus Lp05.

3. An oligopeptide according to claim 1, characterized in that The oligopeptide is synthesized by solid phase synthesis according to the amino acid sequence or obtained by expressing its encoding gene in an engineering strain.

4. The use of the oligopeptide according to claim 1, characterized in that The oligopeptide is used in the modification of lysozyme to enhance the antibacterial spectrum and antibacterial activity.

5. The use of the oligopeptide according to claim 4, characterized in that The oligopeptide is coupled or mixed with natural lysozyme to improve the antibacterial spectrum and antibacterial activity of natural lysozyme.

6. The use of the oligopeptide according to claim 1, characterized in that The oligopeptide is coupled or mixed with natural lysozyme and then used in the preparation of broad-spectrum antibacterial and / or antiseptic products.

7. The use of the oligopeptide according to claim 6, characterized in that The product is a food additive, a feed additive and / or an antibacterial drug.

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