CN109481664B - Application of pediococcus pentosaceus surface protein in inhibition of food-borne pathogenic bacteria - Google Patents
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Abstract
The invention discloses application of pediococcus pentosaceus surface protein in inhibiting food-borne pathogenic bacteria. Compared with the prior art, the extraction method of the Pediococcus pentosaceus surface protein has the advantages of low investment, simple operation and high yield of the antibacterial protein, and the obtained Pediococcus pentosaceus F28-8 surface protein has excellent antibacterial effect on food-borne pathogenic bacteria, thereby providing a new strategy and approach for potential application of the Pediococcus pentosaceus surface protein in the fields of antibacterial agents and antibacterial materials.
Description
Technical Field
The invention belongs to the technical field of new application of pediococcus pentosaceus, and particularly relates to application of pediococcus pentosaceus surface protein in inhibition of food-borne pathogenic bacteria.
Background
Probiotics (Probiotics), also known as microecologics regulator and live bacteria preparation, is a kind of active microorganism which can generate beneficial effect on the physiological function of host, has the functions of regulating intestinal microecology, improving the internal environment of intestinal tract, regulating intestinal flora, inhibiting pathogenic bacteria and the like, and mainly comprises bifidobacterium, lactic acid bacteria and microzyme. Pediococcus pentosaceus (Pediococcus pentosaceus) belongs to the Pediococcus of the Streptococcus family, is gram-positive bacterium, belongs to one of lactic acid bacteria because of being capable of fermenting glucose to generate lactic acid, is widely distributed in traditional fermented foods such as pickles, cheeses, sausages and the like, has an important effect on improving the flavor, nutrition and safety of the fermented foods, and simultaneously has various probiotic functions such as immunoregulation capability, health promotion, pathogenic bacteria invasion resistance and the like.
The various probiotic functions of the lactic acid bacteria are indistinguishable from the presence of their surface active factors. Adhesion is the first step of the probiotic effect of lactic acid bacteria, and the lactic acid bacteria adhere to epithelial cells of host intestines by means of proteins, polysaccharides and the like on the surfaces of the lactic acid bacteria, colonize the epithelial cells, secrete metabolites and the like, and further play important physiological roles of inhibiting adhesion of pathogenic bacteria, inhibiting growth of the pathogenic bacteria and the like. Among the adhesion factors on the surface of lactobacillus, the surface protein of lactobacillus is the main adhesion factor, and the surface protein related to adhesion mainly comprises s-layer protein, primer enzyme Sortase dependent protein, mucosa binding protein, surface protein adhered to extracellular matrix and the like. At present, the research on the structure and function of lactobacillus surface protein is mainly focused on lactobacillus, including mucus binding protein (Mub) of lactobacillus reuteri, collagen binding surface layer protein (Cbsa) and S-layer protein (Slp B) of lactobacillus crispatus, cell wall fixed protein (CwaA) of lactobacillus plantarum NL42, and the like, while relatively few research reports on lactobacillus surface protein are reported.
Disclosure of Invention
The purpose of the invention is as follows: in order to solve the technical problems, the invention provides application of Pediococcus pentosaceus surface protein in inhibiting food-borne pathogenic bacteria, and the surface protein extracted from Pediococcus pentosaceus F28-8 (hereinafter referred to as P.pentosaceus F28-8) has obvious inhibition effect on the food-borne pathogenic bacteria.
The technical scheme is as follows: in order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows:
the application of the surface protein of pediococcus pentosaceus in inhibiting food-borne pathogenic bacteria is disclosed, wherein the surface protein of the pediococcus pentosaceus is extracted from the pediococcus pentosaceus.
Preferably:
the pediococcus pentosaceus is P.pentosaceus F28-8, is preserved in the China general microbiological culture Collection center, has the preservation date of 2014.11.13 and the preservation number of CGMCC No. 9956.
The surface protein extraction method comprises the following steps:
1) taking Pediococcus pentosaceus P.pentacoseus F28-8, inoculating the Pediococcus pentosaceus P.pentacoseus F28-8 into an MRS liquid culture medium according to the inoculation amount of 2-4%, culturing for 12-20h at constant temperature, and centrifugally collecting thalli;
2) adding LiCl solution into the thallus for 0.3-0.7h, centrifuging, discarding the supernatant, re-suspending and mixing with guanidine hydrochloride solution, culturing at constant temperature for 0.5-1.5h, centrifuging, and collecting the supernatant;
3) dialyzing the supernatant obtained in the step 2), centrifuging, taking the supernatant, and freeze-drying to obtain the pediococcus pentosaceus surface protein.
The temperature for the isothermal cultivation in step 1) was 37 ℃.
In the step 2), 10mL of 5mol/L LiCl solution is added into each gram of thallus, and the incubation conditions are as follows: incubating for 20-40 min at 37 ℃; adding 5mL of 4mol/L guanidine hydrochloride solution into each gram of thallus, and culturing at constant temperature under the conditions of: culturing at 37 deg.C for 0.5-3 h.
The molecular interception of the dialysis bag in the step 3) is 14kDa, and the dialysis conditions are as follows: dialyzing in deionized water at 4 ℃ for 48 h.
The pediococcus pentosaceus surface protein is directly used when being applied or is prepared into a microbial inoculum for use.
The invention also provides application of the pediococcus pentosaceus surface protein in preparation of an antibacterial agent capable of preventing and controlling food-borne pathogenic bacteria pollution.
Preferably, the food-borne pathogenic bacteria include gram-positive bacteria (such as staphylococcus aureus and the like) and gram-negative bacteria (such as salmonella and the like).
According to the invention, surface protein is extracted from pediococcus pentosaceus P.pentosaceus F28-8 by a specific method, and experiments prove that the extracted pediococcus pentosaceus surface protein has certain antibacterial performance by acting on food-borne pathogenic bacteria, and is a potential green antibacterial agent which is safer and more reliable in both food industry and medicine industry. The surface protein of pediococcus pentosaceus P.pentosaceus F28-8 is utilized to enhance the inhibition effect on common food-borne pathogenic bacteria in the food industry, and provide information reference for developing a novel green and safe antibacterial agent and expanding the application of lactobacillus surface protein. The method provides a new way and method for producing the antibacterial protein, has the advantages of high separation and extraction rate and low cost, and can be applied to the production of separating and purifying the antibacterial protein from pediococcus pentosaceus on a large scale.
The technical effects are as follows: compared with the prior art, in the application of the method, the extraction method of the pediococcus pentosaceus surface protein is low in investment, simple to operate and high in antibacterial protein yield, and the obtained pediococcus pentosaceus P.pentosaceus F28-8 surface protein is excellent in antibacterial effect on food-borne pathogenic bacteria and provides a theoretical basis for potential application of the pediococcus pentosaceus surface protein in the fields of antibacterial agents and antibacterial materials.
Drawings
FIG. 1 is a growth curve of Pediococcus pentosaceus P.pentosaceus F28-8 and its acid production;
fig. 2 is a transmission electron micrograph of surface protein of pediococcus pentosaceus p.pentosaceus F28-8, wherein a: untreated p.pendosaceus F28-8, arrow: a surface structure; b: LiCl treated p.pendosaceus F28-8;
fig. 3 is SDS-PAGE of pediococcus pentosaceus p.pentasaceus F28-8LiCl extract, wherein M: a standard protein Marker; 1: a surface extract of Pediococcus pentosaceus.
Fig. 4 is a graph of the effect of surface protein of pediococcus pentosaceus p.pentosaceus F28-8 on the growth of salmonella and staphylococcus aureus, wherein (a): the effect of pediococcus pentosaceus surface protein on salmonella growth; (B) the method comprises the following steps The effect of pediococcus pentosaceus surface proteins on the growth of staphylococcus aureus.
Detailed Description
Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated.
The materials and equipment used in the examples were as follows:
1. materials and reagents
P. pentosaceus F28-8, food microbiology laboratory patent preservation strain of the food science and engineering college of the Yangzhou university (China general microbiological culture Collection center, accession number is CGMCC No. 9956); staphylococcus aureus Staphylococcus aureus CICC 22936, Salmonella CICC 21513, purchased from China center for Industrial culture Collection of microorganisms.
Preparing an LB liquid culture medium: tryptone 10 g.L-15 g.L of Yeast extract-1Sodium chloride 10 g. L-1And 1L of distilled water, and carrying out moist heat sterilization at 121 ℃ for 15 min.
2. Apparatus and device
SX-500 model autoclave, TOMMY, Japan; tecnai-12 transmission electron microscope, Phlilps, Netherlands; ALPHAI-2LD PLUS lyophilizer, Christ, Germany; 5800 ultra high resolution time of flight bio mass spectrometer, AB SCIEX, USA.
3. Strain activation method
Inoculating Pediococcus pentosaceus stored in a glycerin tube with the temperature of-20 ℃ into an MRS liquid culture medium according to the inoculation amount of 2% (v/v), culturing at the constant temperature of 37 ℃ for 20h, activating in the MRS liquid culture medium according to the inoculation amount of 2% (v/v), and culturing at the constant temperature of 37 ℃ for 20h for later use.
Inoculating staphylococcus aureus and salmonella stored in a glycerin pipe at the temperature of-20 ℃ into an LB liquid culture medium according to the inoculation amount of 2% (v/v), after carrying out shake culture at the constant temperature of 37 ℃ for 14h, activating the staphylococcus aureus and the salmonella in the LB liquid culture medium according to the inoculation amount of 2% (v/v), and carrying out shake culture at the constant temperature of 37 ℃ for 14h for later use.
Example 1: growth and acid production of Pediococcus pentosaceus
Inoculating-70 deg.C glycerol stock solution at 2% of inoculation amount in MRS culture medium, culturing at 37 deg.C for 24 hr, and measuring OD every 2 hr600nmThe value and the pH value are plotted, and a growth condition and acid production capacity curve chart is drawn.
As shown in FIG. 1, the growth rate of Pediococcus pentosaceus increased gradually with the increase of the culture time and started to enter the logarithmic growth phase at about 4 hours, and reached the stationary phase after 12 hours of culture, and the growth was slowed down. While in the adaptation phase and log phase of Pediococcus pentosaceus, the pH in the medium decreases all the time, from 5.64 to 4.14, and stabilizes at 12 h. The growth of the pediococcus pentosaceus is stable under acidic conditions, which indicates that the pediococcus pentosaceus has stronger acid adaptability and can survive under the condition of lower pH.
Example 2: extraction and analysis of surface protein of pediococcus pentosaceus P.pentosaceus F28-8
(1) The extraction method of the surface protein comprises the following steps:
inoculating the strain into MRS culture medium, culturing at 37 deg.C for 18h, centrifuging at 8000rpm, collecting thallus, washing with PBS, and centrifuging for 2 times. And (3) resuspending the thalli in PBS (phosphate buffer solution), adding 5mol/L LiCl solution (10 mL per gram of thalli) into the thalli of the treatment group, acting at 37 ℃ for 0.5h, centrifuging (8000 r/min, 15min), discarding the supernatant, resuspending and uniformly mixing 4mol/L guanidine hydrochloride solution (5 mL per gram of thalli), treating in a constant-temperature incubator at 37 ℃ for 1h, centrifuging (8000 r/min, 15min), and collecting the supernatant. Transferring the supernatant into a dialysis bag with 14kDa molecular cut-off, dialyzing in deionized water at 4 ℃ for 48h, centrifuging (12000 r/min, 15min), and taking the supernatant, namely the pediococcus pentosaceus surface protein.
(2) Analysis of surface proteins
And (3) suspending thalli before and after LiCl treatment in PBS, respectively dripping the thalli on a special copper net, naturally airing for 10min, dyeing for 2min by using 2% phosphotungstic acid, absorbing excess dye solution by using filter paper, and drying by using a lamp. And (3) putting the sample into a sample stage, and putting the sample into a sample chamber for observation by a Transmission Electron Microscope (TEM). Meanwhile, LiCl extract was analyzed by electrophoresis using SDS-PAGE of 12% separation gel. After the bands on the electrophoresis gel are subjected to gel tapping, PBS is adopted for cleaning, MALDI-TOF-MS mass spectrometry is carried out after trypsin enzymolysis, and the NCBI database is searched by using MASCOT software to obtain protein information.
After the pediococcus pentosaceus F28-8 is cultured at the constant temperature of 37 ℃ for 18h, the result of a transmission electron microscope shows that the cell morphology of the isolated strain is a tetragon and a surface structure exists (figure 2A), and the surface of the strain is basically peeled off after the LiCl treatment (figure 2B). Electrophoresis analysis was performed on the surface protein extracted from LiCl, and as shown in FIG. 3, LiCl extract of Pediococcus pentosaceus showed major protein bands a and b between 45-116kDa in molecular weight.
The bands a and b were separated and recovered by tapping, digested in gel, identified by MALDI-TOF-MS, and compared by MASCOT database search, the band a was identified as N-acetylmuramyl-L-alanine amidase (N-acetylmuramyl-L-alanine amidase) with a theoretical relative molecular weight of 83.5kDa (Table 1). Band b was identified as containing the LysM peptidoglycan-binding protein (LysM peptidoglycan-binding protein) with a theoretical relative molecular weight of 51.1kDa (Table 1).
TABLE 1 protein Mass Spectrometry identification results
Example 3: bacteriostatic application of pediococcus pentosaceus P.pentosaceus F28-8 surface protein
Inoculating activated Staphylococcus aureus (or salmonella) suspension into LB liquid medium, wherein the inoculation amount is 2% (v/v), simultaneously adding Pediococcus pentosaceus surface protein (final concentration is 10 μ g/mL) to experimental group, inoculating Staphylococcus aureus (or salmonella) to control group, performing shake culture at 37 deg.C for 10h, and measuring OD once every 2h600nmThe value is obtained. Drawing (painting)And (5) preparing a growth curve graph. The absorbance value of the experimental group at the same culture time is recorded as ATThe absorbance value of the control group was recorded as ACThe growth inhibition rate of staphylococcus aureus (or salmonella) is calculated according to the formula.
Data statistical analysis: statistical analysis of the data was performed using SPSS 13.0 software, and inter-group significance testing was performed using Duncan's multiplex analysis with significance level P < 0.05.
As shown in FIG. 4, the inhibition rates of Salmonella and Staphylococcus aureus were 31.5% and 15.6% for the treatment group added with the surface protein, respectively, compared to the control group, when the culture was carried out for 10 hours. The result shows that the bacteriostatic effect of the pediococcus pentosaceus surface protein on salmonella is slightly stronger than that of staphylococcus aureus, because other bacteriostatic factors capable of inhibiting gram-negative bacteria exist in the LiCl extract besides bacteriostatic factors capable of effectively inhibiting gram-positive bacteria.
Claims (6)
1. The application of pediococcus pentosaceus surface protein in preparing antibacterial agent capable of preventing and controlling food-borne pathogenic bacteria pollution is characterized in that the pediococcus pentosaceus surface protein is surface protein extracted from pediococcus pentosaceus, and the pediococcus pentosaceus is pediococcus pentosaceusP. pentosaceus F28-8, which is preserved in the China general microbiological culture Collection center with the preservation date of 2014.11.13 and the preservation number of CGMCC No. 9956; the food-borne pathogenic bacteria are staphylococcus aureus or salmonella.
2. Use according to claim 1, characterized in that the surface protein extraction method comprises the following steps:
1) pediococcus pentosaceusP. pentosaceus F28-8, inoculating the strain into an MRS liquid culture medium according to the inoculation amount of 2-4%, culturing at constant temperature for 12-20h, and centrifugally collecting thalli;
2) adding LiCl solution into the thallus for 0.3-0.7h, centrifuging, discarding the supernatant, re-suspending and mixing with guanidine hydrochloride solution, culturing at constant temperature for 0.5-1.5h, centrifuging, and collecting the supernatant;
3) dialyzing the supernatant obtained in the step 2), centrifuging, taking the supernatant, and freeze-drying to obtain the pediococcus pentosaceus surface protein.
3. Use according to claim 2, characterized in that the incubation temperature in step 1) is 37 ℃.
4. The use of claim 2, wherein step 2) comprises adding 10mL of 5mol/L LiCl solution to each gram of thallus, and incubating under the following conditions: incubating for 20-40 min at 37 ℃; adding 5mL of 4mol/L guanidine hydrochloride solution into each gram of thallus, and culturing at constant temperature under the conditions of: culturing at 37 deg.C for 0.5-3 h.
5. The use of claim 2, wherein the molecular cut-off of the dialysis bag in step 3) is 14kDa and the dialysis conditions are: dialyzing in deionized water at 4 ℃ for 48 h.
6. The use of claim 1, wherein the pediococcus pentosaceus surface protein is used directly or as a microbial inoculum.
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| CN111961611A (en) * | 2020-07-21 | 2020-11-20 | 云南省畜牧兽医科学院 | Pediococcus pentosaceus 368 and application thereof |
| CN112011482A (en) * | 2020-08-12 | 2020-12-01 | 扬州大学 | Improved culture method of porcine lactobacillus reuteri |
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