CN115124603A

CN115124603A - Bacteriocin RSQ01 and application thereof

Info

Publication number: CN115124603A
Application number: CN202210747899.8A
Authority: CN
Inventors: 张棋麟; 应建平; 章燕玫; 赵梓舜; 向奕舟
Original assignee: Kunming University of Science and Technology
Current assignee: Kunming University of Science and Technology
Priority date: 2022-06-29
Filing date: 2022-06-29
Publication date: 2022-09-30
Anticipated expiration: 2042-06-29
Also published as: CN115124603B

Abstract

The invention discloses a bacteriocin RSQ01, the amino acid sequence of which is shown as SEQ ID NO:1, the bacteriocin RSQ01 has the advantages of simple structure, safety, low toxicity, easy preparation, wide antibacterial spectrum, high temperature resistance, acid and alkali resistance and the like, and the bacteriocin RSQ01 can be used in the fields of antibacterial drugs, feeds, foods and the like and has wide application prospect.

Description

Bacteriocin RSQ01 and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to bacteriocin RSQ01 and application thereof in preparation of a bacteriostatic agent.

Background

Bacteriocins are a class of ribosomally synthesized polypeptides or proteins produced by Lactic Acid Bacteria (LAB) and have antibacterial activity (Guo et al, 2020). Bacteriocins have many advantages, their efficiency is high, the probability of inducing resistance or non-specific toxicity is low, and they do not form residues. Several bacteriocins in lactic acid bacteria have been reported to have significant antibacterial effects against pathogenic bacteria (Barbosa et al, 2015; Jamaluddin et al, 2017). For example, plantaricin LPL-1 and PA-1 inhibit Listeria monocytogenes (Rodrai guez et al, 2002; Wang et al, 2019) and bacteriocins extracted from Lactobacillus corynebacterium have a significant inhibitory effect on E.coli and Staphylococcus aureus (Yi et al, 2016). Most bacteriocins have broad-spectrum antibacterial properties, and have broad acid, alkali and high temperature resistance (Qin et al, 2019). Unlike traditional antibiotics, which kill bacteria by inhibiting secondary metabolites of the biosynthetic pathway, most bacteriocins kill bacteria by disrupting cellular structures and inducing leakage of cellular contents (Ovchinnikov et al, 2017). This demonstrates that bacteriocins act in a manner that is effective in inhibiting or killing multidrug resistant (MDR) bacteria. In addition, bacteriocins have excellent thermal and storage stability, and compared to conventional antibiotics, have a wide range of applications, and compared to chemically synthesized conventional food preservatives, natural, highly efficient bacteriocins are safe and non-toxic to human hosts (L' opez-Cuellar et al, 2016). Shows good development and application potential in the fields of food, biological control, medical treatment and the like (Aslam et al, 2020; Qin et al, 2019). LAB-produced bacteriocins have great potential in biological preservation due to their generally accepted safety (GRAS), but currently only pediocin PA-1 and nisin (nisin) have been commercialized as food preservatives (Delves Broughton et al, 2005; Simha et al, 2012), and the rest have been limited to experimental studies.

Different bacteriocins have different bacteriostatic efficiency and bacteriostatic property, and the same bacteriocin has different bacteriostatic activity under different conditions, and different external environments can also influence the structure, the property and the bacteriostatic effect of the bacteriocin. Therefore, the search for novel bacteriocins and the research on the bacteriostatic stability of the bacteriocins are of great significance to the subsequent research and application of the bacteriocins.

Disclosure of Invention

The invention provides a bacteriocin RSQ01, which is obtained by separating Lactococcus lactis (Lactococcus lactis) from yellow river firefly intestinal tracts, and the amino acid sequence of the bacteriocin RSQ01 is shown as SEQ ID NO: 1.

Another object of the present invention is to use the bacteriocin RSQ01 described above in the preparation of bacteriostatic agents against bacterial species including Staphylococcus aureus (Staphylococcus aureus), Listeria monocytogenes (Listeria monocytogenes), Salmonella enteritidis (Salmonella enteritidis), Salmonella choleraesuis (Salmonella choleraesuis), Escherichia coli (Escherichia coli), Enterobacter sakazakii (Enterobacter sakazakii), and Pseudomonas aeruginosa (Pseudomonas aeruginosa).

The purpose of the invention is realized by the technical scheme that:

1. lactococcus lactis isolated from the intestine of yellow firefly that produces bacteriocin;

2. culturing lactococcus lactis with MRS culture medium, centrifuging to obtain supernatant, filtering with membrane, and using

A pure protein separation and purification platform (GE, Sweden, USA) for separating and purifying to obtain small molecule peptide RSQ 01;

3. determining the molecular weight and the amino acid sequence of the small molecular peptide RSQ 01;

4. and (3) detecting the bacteriostatic activity and stability of the bacteriocin RSQ 01.

The invention has the following beneficial effects:

the bacteriocin RSQ01 has the advantages of simple structure, safety, low toxicity, easy preparation, wide antibacterial spectrum, high temperature resistance, acid and alkali resistance and the like. The bacteriocin RSQ01 can be used in the fields of antibacterial drugs, feeds, foods and the like, and has a wide application prospect.

Drawings

FIG. 1 shows that of small molecule peptide RSQ01

Detecting a spectrogram in a purification process;

FIG. 2 is a graph showing the effect of small molecule peptide RSQ01 on Salmonella enteritidis morphology, wherein A is a control group and B is a treatment group;

FIG. 3 is a primary mass spectrum of small molecule peptide RSQ 01;

FIG. 4 is a secondary mass spectrum of small molecule peptide RSQ 01;

FIG. 5 shows the results of experiments on the resistance of bacteriocin RSQ01 to various temperatures;

FIG. 6 shows the results of experiments on the resistance of bacteriocin RSQ01 to various pH values;

FIG. 7 shows the results of experiments with bacteriocin RSQ01 resistant to different enzymes.

Detailed Description

The technical scheme of the invention is further described in detail by the following examples, but the content of the invention is not limited to the following examples, the method in the examples is a conventional method unless otherwise specified, and the materials, reagents and the like used are obtained from commercial sources unless otherwise specified; the methods used in the following examples are conventional methods unless otherwise specified, and the percentage concentrations are mass/volume (w/v) percentage concentrations unless otherwise specified; the low temperature centrifugation in the following examples, if not noted, was performed at 4 ℃;

flavobacterium reichersonii was bred at the institute of Life sciences, Kunming technology university, Yunnan, China, and lactococcus lactis was cultured in MRS medium at 37 deg.C, and the culture medium of the strain used in the bacteriostatic experiments is shown in Table 1 and cultured at 37 deg.C. All bacteria were stored at-80 ℃ in culture medium containing 20% glycerol (v/v).

Example 1: bacteriocin-producing lactococcus lactis isolation

(1) Mix the enteric canal of Luciola retrei with sterile 0.85% NaCl solution (100mL) and then use the sterile 0.85% NaCl solution to dilute the mixture to 10 gradient ^-7 Take 10 out ^-1 、10 ^-3 、10 ^-5 、10 ^-7 Respectively coating 100 mu L of 4 gradient bacterial liquids on an MRS culture medium, setting 3 times of each concentration, uniformly coating by using a flat plate coating method, and then culturing the MRS solid culture medium at 37 ℃ for 24 hours;

(2) selecting single colonies in the MRS solid culture medium, respectively inoculating the single colonies in 5mL of MRS liquid culture medium, and performing shake culture at a constant temperature of 37 ℃ for 24 hours;

(3) centrifuging MRS liquid culture medium of different single colonies at 4 deg.C for 10min (8000g), collecting supernatant, and filtering with filter with pore diameter of 0.22 μm to obtain cell-free supernatant containing no bacteria; adding 200 mu L of supernatant into sterilized Oxford cup (inner diameter is 6mm), placing on double-layer flat plate containing Salmonella enteritidis and Staphylococcus aureus, culturing at 37 deg.C in constant temperature incubator for 24h, and observing the size of zone to determine antibacterial activity, wherein the diameter of the zone is measured by vernier caliper;

(4) selecting a strain with the largest inhibition zone, performing species identification on the bacteria by using a bacteria 16S rDNA sequence sequencing method, and determining the strain to be lactococcus lactis by combining morphological characteristics;

(5) in addition, the method in the step (3) is used for further determining the bacteriostatic activity of the cell-free supernatant of the lactococcus lactis liquid culture medium on escherichia coli, listeria monocytogenes and salmonella choleraesuis, and the result shows that the strain also has bacteriostatic activity on the 3 pathogenic bacteria, and the bacteriostatic circle on escherichia coli is 18.56 +/-0.13 mm, so that the lactococcus lactis strain with wide antibacterial spectrum and high antibacterial activity is obtained.

Example 2: separation, purification and identification of small molecule peptide RSQ01

1. EXAMPLE 1 isolated lactococcus lactis was cultured to OD in 100mL of MRS liquid medium ₅₉₅ 0.3-0.6, inoculating the strain into a 1L MRS liquid culture medium in an inoculation amount of 0.5%, and culturing at 37 ℃ for 24 h;

2. centrifuging at 8000g at 4 deg.C for 10min to remove bacterial cells, and collecting supernatant;

3. filtering the supernatant obtained in the step (2) by using a filter membrane with the pore diameter of 0.22 mu m to obtain cell-free supernatant;

4. use of

The pure protein separation and purification platform (GE, Sweden, USA) obtains the small molecule peptide RSQ01

Equilibrating Superdex with 20mmol/L phosphate buffer (pH5.2) ^TM After 30 increate 10/300GL chromatography column, cell-free supernatant was loaded into the column using the following operating parameters: equilibrium volume was 2 Column Volumes (CV), eluent pH 5.3 imidazole, elution volume was 1.5CV, flow rateIs 0.5 mL/min; monitoring the change of the absorbance value of the eluent under UV 280nm, collecting 0.5mL of the eluent under 4 main peaks of A1, A2, A3 and A4 respectively, collecting the eluent in a 15mL tube, and measuring the bacteriostatic activity of the A1, A2, A3 and A4 peak collectors on salmonella enteritidis by adopting an Oxford cup double-layer plate method, wherein the result of a bacteriostatic zone shows that the size of the bacteriostatic zone of the A1 peak collector is 20.15 +/-0.42 mm and has the bacteriostatic activity (figure 1A);

can be reused

Purifying the A1 peak collection by a pure protein separation and purification platform, and setting the parameters as above; re-purifying the A1 peak to obtain a B1 single peak (figure 1B), and determining the bacteriostatic activity of the B1 peak collector on salmonella enteritidis by using an Oxford cup double-layer plate method, wherein the size of a bacteriostatic circle is 24.30 +/-0.23 mm; the result proves that the compound is a single substance and has good bacteriostatic activity, and the compound is named as RSQ 01;

5. the concentration of RSQ01 was determined by using a bicinchoninic acid (BCA) kit (bisharp, Beijing, China), according to the kit instructions, followed by lyophilization in an FD-2 lyophilizer and storage;

6. influence of small molecule peptide RSQ01 on Salmonella enteritidis morphology

Observing the integrity and morphological characteristics of cell membranes of the salmonella enteritidis treated by the small molecular peptide RSQ01 by using a scanning electron microscope to evaluate the influence degree of indicator bacterium planktonic cells after the RSQ01 treatment

2mL (10) was added to the tube ⁷ CFU/mL), centrifuging at 4 deg.C and 8000g for 5min, taking the thallus precipitated in EP tube, resuspending with PBS buffer solution, and repeating for three times; subsequently, the small molecule peptide RSQ01 (11.63. mu.g/mL) was added and incubated at 37 ℃ for 60 min; then 2mL of 2.5% glutaraldehyde is added, and the mixture is fixed for 12 hours at 4 ℃; washing with PBS buffer solution, dehydrating under ethanol concentration gradient (30%, 50%, 60%, 70%, 80%, 90%, 100%), then dehydrating in a freeze dryer overnight, adhering the dehydrated bacterial cells on a polished silicon wafer, drying, and spraying gold; detection under a FlexSEM1000 scanning electron microscope; as a result, theReferring to fig. 2, it can be seen that the small molecule peptide RSQ01 can destroy the cell membrane of salmonella enteritidis, change the cell morphology of the bacteria, and thus achieve the bacteriostatic effect.

6. Molecular weight of small molecule peptide RSQ01

The molecular weight and the amino acid sequence of the small molecular peptide RSQ01 are determined by combining nano liquid chromatography with tandem mass spectrometry (nanolC-MS/MS)

Dissolving the purified small-molecule peptide RSQ01 in ddH ₂ O, reduced with 10mmol/L dithiothreitol sugar at 26 ℃ for 1h, then aminated with 20mmol/L acetamide iodide at room temperature for 40min, lyophilized, resuspended in 20. mu.L of 0.1% formic acid before chromatography, and the peptide fragment analysis was performed in a Multimate 3000 system equipped with QOxctive ^TM Hybrid quadrupole-Orbitrap ^TM Mass spectrometer, and prepare ESI nanometer spray source; a reverse phase nano-chromatographic column with a built-in volume of 150 μm × 15cm was used, and Rerosil-PurC 18-AQ 1.9 μm resin was used as the column

Filling; mobile phase a consisted of ultra pure water containing 0.1% formic acid and mobile phase B consisted of acetonitrile containing 0.1% formic acid. The linear gradient elution of acetonitrile was as follows: from 6% to 9% mobile phase B was used for 5 min; from 9% to 14% mobile phase B was used for 15 min; from 14% to 30% mobile phase B was used for 30 min; from 30% to 40% of mobile phase B was used for 8min, from 40% to 95% of mobile phase B was used for 2min, and the flow rates were all 0.6. mu.L/min. 5 μ L of sample was loaded into the system; mass spectrometry analysis used a single full scan (MS) with the following parameters: resolution in the range of 100-1,500m/z, 400m/z is 70,000; then 10 data dependent scans (MS/MS) were performed. Data-dependent scans of mass spectrometry measurements were performed using Xcalibur 2.1.2 software in orbitrap (using a spray voltage of 2.2kV and a capillary temperature of 270 ℃); protein identification was performed using PeaksStudio 8.5(Bioinfirmatics solutions Inc., Waterloo, ONT, Canada), with a primary mass spectrum shown in FIG. 3 and a secondary mass spectrum shown in FIG. 4.

The result shows that MALDI-TOF-MS shows that the molecular weight of the small molecule peptide RSQ01 is 1901.87Da, and the amino acid sequence of the small molecule peptide RSQ01 is shown in SEQ ID NO. 1 according to the complete genome sequence and molecular weight analysis.

Using proteomic BLAST against GenBank (www.ncbi.nlm.nih.gov/BLAST), bacteriocin RSQ01 showed no homology to the reported bacteriocins. In addition, the alignment of other mature class II bacteriocins revealed that bacteriocin RSQ01 is a novel bacteriocin. Therefore, the bacteriocin RSQ01 from lactococcus lactis is a novel class IIa bacteriocin.

Experimental example 3 bacteriocinRSQ01Functional characterization of

1. BacteriocinRSQ01Antibacterial spectrum of

The bacteriocin purified in example 2 was addedRSQ01For determining the antibiogram, an antibiogram against indicator strains containing food spoilage bacteria and food-borne pathogens was tested and the results are shown in table 1:

TABLE 1

；

In the table: ^b diameter (mm) of the antibacterial ring: +++:>20mm； ++：11-20mm；+：5-10mm。

2. stability of bacteriocin RSQ01

To evaluate the stability and protein properties of bacteriocin RSQ01, its thermostability, acid-base resistance, storage stability and enzyme sensitivity were examined.

The evaluation of the heat stability of bacteriocin RSQ01 was carried out by dissolving bacteriocin lyophilized powder in PBS buffer (pH7.4) to a final concentration of 11.63. mu.g/mL, treating at 60 ℃, 80 ℃ and 100 ℃ for 30min, respectively, while using bacteriocin at room temperature (25 ℃) as a control; the antibacterial activity of the bacteriocin on staphylococcus aureus after treatment at different temperatures is detected by an oxford cup double-layer plate method, and the result is shown in figure 5;

dissolving the bacteriocin freeze-dried powder in PBS buffer solutions with different pH values (2, 4, 6, 8 and 10), culturing for 1h at 37, detecting the bacteriostatic activity of the bacteriocin on staphylococcus aureus after treatment at different temperatures by using an Oxford cup double-layer plate method with the original pH value of the bacteriocin being 4.5 as a control, and obtaining a result shown in figure 6;

detecting sensitivity of bacteriocin (11.63 μ g/mL) to various enzymes, adding enzyme to bacteriocin solution to a final concentration of 1mg/mL, incubating at 37 ℃ for 2h, and then inactivating at 80 ℃ for 10min, wherein the bacteriocin solution without enzyme is used as a positive control, and the enzymes are trypsin, alpha-amylase, beta-amylase, proteinase K, pepsin, lipase and catalase respectively; the bacteriostatic activity of the bacteriocin on staphylococcus aureus after treatment at different temperatures was detected by the oxford cup double-layer plate method, and the result is shown in fig. 7.

The results show that bacteriocin RSQ01 shows good tolerance to high temperature, acid, alkali and enzyme.

Sequence listing

<110> university of Kunming science

<120> bacteriocin RSQ01 and application thereof

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 20

<212> PRT

<213> Lactococcus lactis (Lactococcus lactis)

<400> 1

Gly Thr Pro Ser Gly Gly Ala Asp Gly Ile Gly Phe Ala Phe His Pro

1 5 10 15

Glu Glu Val Gly

20

Claims

1. A bacteriocin RSQ01 has an amino acid sequence shown in SEQ ID NO. 1.

2. The use of the bacteriocin RSQ01 of claim 1 for the preparation of a bacteriostatic agent.

3. Use according to claim 2, characterized in that: bacteriostatic agents inhibit Staphylococcus aureus (Staphylococcus aureus), Listeria monocytogenes (Listeria monocytogenes), Salmonella enteritidis (Salmonella enteritidis), Salmonella choleraesuis (Salmonella choleraesuis), Escherichia coli (Escherichia coli), Enterobacter sakazakii (Enterobacter sakazakii), Pseudomonas aeruginosa (Pseudomonas aeruginosa).