CN114231462A - Active strain for inhibiting drug-resistant food-borne pathogenic bacteria and application thereof - Google Patents
Active strain for inhibiting drug-resistant food-borne pathogenic bacteria and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of microorganisms, and particularly discloses an active strain for inhibiting drug-resistant food-borne pathogenic bacteria and application thereof. The 16S rDNA sequence of the active strain for inhibiting the drug-resistant food-borne pathogenic bacteria is shown as SEQ ID NO. 1. The active strain is Pseudomonas fluorescens (Pseudomonas fluorescens) which is preserved in Guangdong province microorganism strain preservation center with the preservation number as follows: GDMCC No: 61981. the antibacterial active substance metabolized by Pseudomonas fluorescens has broad-spectrum antibacterial activity, especially has strong antibacterial activity on drug-resistant staphylococcus aureus, and can be used as a potential food preservative to be applied to food preservation.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to an active strain for inhibiting drug-resistant food-borne pathogenic bacteria and application thereof.
Background
Food safety is the basis of harmony, stability and development progress of human society. China clearly puts forward a development direction for comprehensively building healthy China in the future, and attaches importance to food safety to become a focus of social attention. At present, food safety problems frequently occur in the global scope, food-borne diseases caused by microorganisms are recognized as the first food safety problems, 70% of children who die from food poisoning are caused by pathogenic microorganisms, food spoilage caused by annual microbial pollution accounts for more than 10% of food production value, and prevention and control of pathogenic microorganisms are important requirements for food safety. Food preservatives are the most direct and effective way to control the growth and reproduction of various microorganisms in food products.
The chemical preservative has many disadvantages caused by long-term use, such as influence on the original quality of food and damage to human health, so that the effect and safety of the food preservative are questioned. At the same time, the abusive use of antibiotics makes drug-resistant strains in food a great risk to human health. For example, Staphylococcus aureus (Staphylococcus aureus) among food-borne pathogenic bacteria can cause various diseases such as pneumonia, enteritis, septicemia and the like, is a main pathogenic bacterium causing food poisoning, and food which is easily contaminated by Staphylococcus aureus includes meat, eggs, milk products and the like. Therefore, the development of highly effective, safe and healthy natural preservatives has been a hot point of research in the food industry.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide an active strain for efficiently inhibiting drug-resistant food-borne pathogenic bacteria and application thereof. The active strain is Pseudomonas fluorescens (Pseudomonas fluorescens), and the metabolized antibacterial active substance of the strain has broad-spectrum antibacterial activity, especially has strong antibacterial activity on drug-resistant staphylococcus aureus, and can be used as a potential food preservative to be applied to food preservation.
In order to achieve the purpose, the invention adopts the technical scheme that:
the invention provides an active strain for inhibiting drug-resistant food-borne pathogenic bacteria, wherein the 16S rDNA sequence of the active strain is shown as SEQ ID NO. 1.
The nucleotide sequence of 16S rDNA of the active strain was highly similar to Pseudomonas fluorescens (Pseudomonas fluorescens) with a similarity of 100% when analyzed by alignment with BLAST in NCBI database.
As a preferred embodiment of the active strain of the present invention, the active strain is Pseudomonas fluorescens (Pseudomonas fluorescens) S-541, which is deposited at the Guangdong province collection of microorganisms at 10 months and 27 days 2021, and the deposit number is GDMCC No: 61981.
the invention also provides the application of the active bacterial strain in preparing antibacterial active substances.
As a preferred embodiment of the use according to the invention, the antibacterial active substance is ricinoleic acid.
Metabolites (antibacterial active substances) generated after the metabolism of Pseudomonas fluorescens (Pseudomonas fluorescens) S-541 have broad-spectrum antibacterial activity, especially have strong antibacterial activity on drug-resistant staphylococcus aureus, and can be used as a potential food preservative to be applied to food preservation.
Preferably, Pseudomonas fluorescens (Pseudomonas fluorescens) S-541 is cultured with MH solid culture medium for 2-4 days, and is subjected to methanol solvent extraction and reduced pressure concentration to obtain a crude extract of the antibacterial active substance. And further adopting antibacterial activity tracking, separating and purifying the crude extract of the strain by utilizing semi-preparative liquid chromatography to obtain a pure compound, preliminarily determining that the active compound of the strain is ricinoleic acid through high-resolution mass spectrometry and database comparison, wherein the compound not only has stronger inhibiting effect on gram-positive bacteria such as staphylococcus aureus, listeria monocytogenes and the like, but also has inhibiting effect on gram-negative bacteria such as side escherichia coli and salmonella, and shows good antibacterial activity on drug-resistant strains of the strains.
The invention also provides the application of the active strain in inhibiting food-borne pathogenic strains and corresponding drug-resistant pathogenic strains.
As a preferred embodiment of the application of the invention, the food-borne pathogenic strains comprise Escherichia coli (Escherichia coli) CMCC44102, Salmonella typhimurium (Salmonella typhimurium) CMCC26003, Staphylococcus aureus (Staphylococcus aureus) GIM 1.644, Listeria monocytogenes (Listeria monocytogenes) GIM 1.347, and the drug-resistant pathogenic strains comprise drug-resistant Escherichia coli 2624-2, drug-resistant Salmonella 54-9, drug-resistant Staphylococcus aureus 117-2 and drug-resistant Listeria monocytogenes 1846-1.
According to the invention, an Oxford cup method is adopted to test the bacteriostatic activity of Pseudomonas fluorescens (Pseudomonas fluorescens) S-541, the food-borne pathogenic bacterial strain and the corresponding drug-resistant bacterial strain are selected as indicator bacteria, and the experimental result shows that the metabolite of the Pseudomonas fluorescens (Pseudomonas fluorescens) S-541 has broad-spectrum bacteriostatic activity, detects better antibacterial activity on the indicator bacteria, and especially has strong bacteriostatic effect on drug-resistant staphylococcus aureus.
In a fourth object, the invention provides a fermentation supernatant, which comprises the active strain for inhibiting drug-resistant food-borne pathogenic bacteria.
Inoculating Pseudomonas fluorescens (Pseudomonas fluorescens) S-541 into MH liquid culture medium, culturing at 37 deg.C for 36-48h, and centrifuging to obtain fermentation supernatant with antibacterial activity.
More preferably, Pseudomonas fluorescens (Pseudomonas fluorescens) S-541 is inoculated into MH liquid culture medium, the MH liquid culture medium is placed in a constant temperature shaking table to be cultured at 37 ℃ and 160r/min, after 2 generations of activation, the MH liquid culture medium is inoculated into 1L MH liquid culture medium according to the inoculum size of 10 percent, the MH liquid culture medium is cultured for 48h at 37 ℃, fermentation liquor is collected and centrifuged for 10min at 8000r/min and 4 ℃, fermentation supernatant is reserved, and the fermentation supernatant is filtered by using a 0.22 mu m filter membrane to obtain fermentation supernatant with bacteriostatic activity.
The fifth purpose of the invention is to provide the application of the fermentation supernatant in inhibiting drug-resistant food-borne pathogenic bacteria. More preferably, the drug-resistant food-borne pathogenic bacterium is drug-resistant staphylococcus aureus 117-2.
The active compound in the fermentation supernatant containing the active strain can destroy the cell wall and the cell membrane of the drug-resistant staphylococcus aureus 117-2, so that the cell contents are leaked, the thallus is shriveled and shrunk, and the growth of the drug-resistant staphylococcus aureus 117-2 is inhibited.
The sixth purpose of the invention is to provide the application of the active bacterial strain for inhibiting drug-resistant food-borne pathogenic bacteria in the preparation of food preservatives.
According to a seventh object, the invention provides a food preservative comprising the active strain.
The Pseudomonas fluorescens (Pseudomonas fluorescens) S-541 obtained by separation is an active strain with broad-spectrum bacteriostasis and high-efficiency inhibition on drug-resistant food-borne pathogenic bacteria, and has the development value of potential natural microbial food preservatives.
Compared with the prior art, the invention has the following beneficial effects:
the invention separates a Pseudomonas fluorescens (Pseudomonas fluorescens) S-541 with high-efficiency inhibition of drug-resistant food-borne pathogenic bacteria from the vegetable garden soil of the white cloud area in Guangdong Guangzhou city, and the result of the antibacterial activity test shows that the metabolite of the Pseudomonas fluorescens has broad-spectrum antibacterial activity, especially has strong antibacterial effect on drug-resistant staphylococcus aureus, and can be used as a potential food preservative to be applied to food preservation. The invention utilizes solvent extraction and chromatographic purification technology to separate and purify the crude extract of the strain to obtain a pure compound, and the active substance of the strain is determined to be the compound ricinoleic acid through high-resolution mass spectrometry and database comparison.
Drawings
FIG. 1 is a phylogenetic tree constructed by phylogenetic analysis of Pseudomonas fluorescens S-541;
FIG. 2 is a graph showing the results of testing the bacteriostatic effect of the fermentation supernatant of Pseudomonas fluorescens (Pseudomonas fluorescens) S-541 on drug-resistant Staphylococcus aureus 117-2 by Oxford cup method;
FIG. 3 is a product analysis map of a crude extract of Pseudomonas fluorescens S-541 using semi-preparative chromatography;
FIG. 4 is an analytical map of high performance liquid chromatography for Compound A;
FIG. 5 is an analysis mass spectrum of the ultra-high resolution mass spectrum on the compound A;
FIG. 6 is a molecular structure diagram of the compound ricinoleic acid;
FIG. 7 is a diagram of the bacteriostatic effect of the fermentation supernatant of Pseudomonas fluorescens (Pseudomonas fluorescens) S-541 on drug-resistant Staphylococcus aureus 117-2 under a scanning electron microscope at a resolution of 5nm (FIG. A is a control group treated by distilled water, and FIG. B, C, D, E is samples treated for 1h, 3h, 6h and 12h, respectively).
Detailed Description
To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments.
In the following examples, the experimental methods used were all conventional methods unless otherwise specified, and the materials, reagents and the like used were commercially available without otherwise specified.
The indicator strains mentioned in the following examples were provided by the Guangdong microbiological Culture Collection Center (Guangdong microbiological Culture Collection Center), and the test materials used were purchased from conventional Biochemical agents, unless otherwise specified.
Example 1 16S rDNA sequence analysis and construction of phylogenetic Tree of Pseudomonas fluorescens S-541
The invention separates an active strain from vegetable garden soil of a white cloud area in Guangdong Guangzhou city as a strain to be tested, and extracts DNA of the active strain.
Extracting total DNA of the strain: scraping a proper amount of strain to be detected from a NA slant culture medium preserved at low temperature (4 ℃) in a laboratory, inoculating the strain to a 100ml liquid culture medium, culturing the strain at 28 ℃ for 72 hours, extracting genomic DNA by using a bacterial genomic DNA extraction kit (BIOMIGA, America), performing the operation steps according to the kit specification, and detecting the extraction effect of the obtained genome on a gel imager after 0.8% agarose electrophoresis and performing genome sequencing.
Cloning primers for gene sequence determination of active strain 16S rDNA sequence bacterial universal primers 27f (5'-GTGCTGCAGAGAGTTTGATCCTGGCTCAG-3') and 1492r (5'-CACGGATCCTACGGGTACCTTGTTACGACTT-3') were used. Setting the reaction condition parameters in a PCR amplification instrument as follows: pre-denaturation at 95 deg.C for 3min, denaturation at 94 deg.C for 1min, renaturation at 55 deg.C for 40s, extension at 72 deg.C for 2 min, 30 cycles, constant temperature at 72 deg.C for 10min, and termination at 22 deg.C. Transferring the PCR reaction product to Huada gene sequencing to obtain the 16S rDNA sequence of the strain. The 16S rDNA sequence of the strain is shown in SEQ ID NO. 1.
Comparing the 16S rDNA sequence of the active strain with the 16S rDNA sequence of a known related strain in an NCBI online database in a BLAST mode, downloading the 16S rDNA sequence of the related strain of the strain through the database, performing multiple comparison by using Clustal X1.8 software, performing cluster analysis by using Mega 8.0, setting parameters for 1000 times Bootstrap by using an adjacency method (N-J) method, constructing a phylogenetic tree for the active strain, and analyzing the phylogenetic status of the active strain.
Phylogenetic analysis of the strains of the present invention the phylogenetic tree constructed by reference to fig. 1 (the neighborhood phylogenetic tree constructed based on the 16S rDNA gene sequence shows the relationship between the active strain and the closely related strains, and the GenBank accession number of the corresponding strain is shown in parentheses.) shows that the active strain is highly similar to Pseudomonas fluorescens (Pseudomonas fluorescens), with a similarity of 100%. The invention discloses an active strain named as Pseudomonas fluorescens (Pseudomonas fluorescens) S-541 which is preserved in Guangdong province microorganism strain preservation center at 2021, 10 months and 27 days, and the address is as follows: china Guangzhou city, Jielizhou No. 100, Guangdong province microbial research institute, the preservation number is GDMCC No: 61981.
example 2 determination of the antibacterial Activity of the fermentation supernatant of Pseudomonas fluorescens S-541
1) Obtaining of fermentation supernatant of Pseudomonas fluorescens (Pseudomonas fluorescens) S-541:
pseudomonas fluorescens (Pseudomonas fluorescens) S-541 preserved on an NA agar slant is inoculated in an NB liquid culture medium for activation and shake culture at 37 ℃ and 160r/min for 24 h. An appropriate amount of activated Pseudomonas fluorescens (Pseudomonas fluorescens) S-541 is taken out and inoculated into 100mL of MH liquid culture medium according to the inoculation amount of 10 percent, and shaking cultured for 72h at 37 ℃ and 160r/min for shaking culture. And centrifuging the cultured strain fermentation liquor at 8000r/min at 4 ℃ for 10min to obtain fermentation supernatant.
2) Antibacterial activity test of Pseudomonas fluorescens (Pseudomonas fluorescens) S-541 fermentation supernatant:
taking food-borne pathogenic bacteria and corresponding drug-resistant strains as indicator bacteria, firstly preparing the indicator bacteriaPouring a proper amount of NA solid culture medium which is cooled after sterilization into an aseptic culture dish in an aseptic operation platform, coating 100 mu L of indicator bacterium liquid after the culture medium is cooled and solidified, and enabling the initial concentration of the bacterium liquid to be 107cfu/mL. And clamping the sterilized Oxford cup by using a pair of tweezers, placing the Oxford cup on a culture dish coated with the indicator bacteria, taking 100 mu L of fermentation supernatant by using a pipette, adding the fermentation supernatant into the Oxford cup, and recording the serial number. And (3) placing the rescreened culture dish into a constant-temperature incubator for culture, and placing the bacteria indicator bacteria at 37 ℃ for culture for 24 h. Observing the size of the inhibition zone around the oxford cup on the culture plate, measuring the diameter of the inhibition zone by using a digital caliper and recording, wherein the figure 2 shows the inhibition effect of the supernatant obtained by fermenting Pseudomonas fluorescens (Pseudomonas fluorescens) S-541 by adopting an oxford cup method on the drug-resistant staphylococcus aureus 117-2.
Table 1 shows the information of the indicator bacteria and the corresponding drug-resistant strains, and the results of the antibacterial activity of the fermentation supernatant of Pseudomonas fluorescens S-541 on the indicator bacteria.
TABLE 1
Note: g +, gram positive bacteria; g-is gram-negative bacteria; the antibiotic-resistant medicines are: ampicillin (AMP), Amoxicillin (AMC), Ceftazidime (CAZ), Cefotaxime (CTX), cephalosporin (KF), cefoxitin (FOX), Nalidixic Acid (NA), Ciprofloxacin (CIP) S, Norfloxacin (NOR), gentamicin (CN), chloramphenicol (C), compound Sulfamethoxazole (SXT), streptomycin (S), kanamycin (K), Tetracycline (TE), erythromycin (E), clindamycin (DA), Rifampicin (RD), Doxycycline (DO), penicillin (P).
Example 3 extraction, isolation and characterization of the active product of Pseudomonas fluorescens S-541
Culturing Pseudomonas fluorescens (Pseudomonas fluorescens) S-541 with MH solid culture medium for 2-4 days, extracting with methanol for 24h, and concentrating under reduced pressure at 40 deg.C with rotary evaporator to obtain crude extract containing antibacterial active substances. Dissolving the crude extract in 5ml methanol, filtering with 0.22 μm membrane, and separating and purifying by semi-preparative liquid chromatography under the following conditions: shimadzu LC-20A, column: waters C18 column (20 mm. times.250 mm, 5 μm) with volume fraction of 15% methanol and ultrapure water as mobile phase at a flow rate of 3mL/min to give two fractions of fractions A and B as shown in FIG. 3.
By tracing the antibacterial activity, component a shows good antibacterial activity, and therefore, component a is further purified in a semi-preparative chromatography using the same method to obtain the final purified compound a, as shown in fig. 4.
The purified compound A was subjected to ultra high resolution mass spectrometry as shown in FIG. 5, in [ M + Na ]]+ and [ M + H]The corresponding molecular weights were measured in + ion mode: 321.23972Da and 299.25784Da, giving compound A a molecular weight: 298.25071Da, structural formula: c18H34O3The active substance of the strain is preliminarily determined to be a compound ricinoleic acid, the molecular structure of the compound is shown in figure 6, the compound has a strong inhibiting effect on gram-positive bacteria such as staphylococcus aureus, listeria monocytogenes and the like, simultaneously has an inhibiting effect on gram-negative bacteria such as side escherichia coli and salmonella, and shows good antibacterial activity on drug-resistant strains of the strains.
Example 4 bacteriostatic mechanism of Pseudomonas fluorescens S-541
Centrifuging drug-resistant Staphylococcus aureus 117-2 in logarithmic growth phase at 3500r/min for 10min, discarding supernatant, washing with sterile buffer solution for 3 times, and diluting to total colony count of 106Adding 1 volume of Pseudomonas fluorescens (Pseudomonas fluorescens) S-541 fermentation supernatant into CFU/ml bacterial suspension, taking distilled water with the same volume as a blank control group, culturing at 37 ℃ and 150r/min, and sampling for 1h, 3h, 6h and 12h respectively to be used as samples to be detected. Preparing centrifugal sample to be tested from scanning electron microscope sample, buffering thallus deposit with phosphoric acidWashing with solution for 3 times, fixing with 2.5% glutaraldehyde for 2 hr, dehydrating with ethanol gradient, coating onto metal foil, vacuum drying, fixing, spraying gold, observing morphological structure change under scanning electron microscope, and treating with distilled water as control group.
As shown in FIG. 7, panel A is the control treated with distilled water, and panel B, C, D, E is the samples treated for 1h, 3h, 6h and 12h, respectively. The results show that the active compound in the fermentation supernatant of Pseudomonas fluorescens (Pseudomonas fluorescens) S-541 can destroy the cell wall and the cell membrane of the drug-resistant staphylococcus aureus 117-2, cause the leakage of the cell content, and cause the bacterial body to be shriveled and shriveled, thereby inhibiting the growth of the strain.
Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
SEQUENCE LISTING
<110> institute of microbiology, academy of sciences of Guangdong province (center for microbiological analysis and detection of Guangdong province)
<120> active strain for inhibiting drug-resistant food-borne pathogenic bacteria and application thereof
<130> 20211210
<160> 1
<170> PatentIn version 3.5
<210> 1
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<212> DNA
<213> Artificial Synthesis
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Claims (10)
1. An active strain for inhibiting drug-resistant food-borne pathogenic bacteria is characterized in that the 16S rDNA sequence of the active strain is shown as SEQ ID NO. 1.
2. The active strain according to claim 1, wherein the active strain is Pseudomonas fluorescens (Pseudomonas fluorescens) deposited at the Guangdong provincial collection of microorganisms with the deposit number GDMCC No: 61981.
3. use of an active strain according to claim 1 or 2 for the preparation of an antibacterially active substance.
4. Use according to claim 3, wherein the antibacterial active substance is ricinoleic acid.
5. Use of an active strain according to claim 1 or 2 for inhibiting food-borne pathogenic strains and the corresponding drug-resistant pathogenic strains.
6. The use of claim 5, wherein the food-borne pathogenic strains comprise Escherichia coli (Escherichia coli) CMCC44102, Salmonella typhimurium (Salmonella typhimurium) CMCC26003, Staphylococcus aureus (Staphylococcus aureus) GIM 1.644, Listeria monocytogenes (Listeria monocytogenes) GIM 1.347, and the drug-resistant pathogenic strains comprise drug-resistant Escherichia coli 2624-2, drug-resistant Salmonella 54-9, drug-resistant Staphylococcus aureus 117-2, and drug-resistant Listeria monocytogenes 1846-1.
7. A fermentation supernatant comprising the active strain of claim 1 or 2 for inhibiting drug-resistant food-borne pathogenic bacteria.
8. Use of the fermentation supernatant of claim 7 for the inhibition of drug-resistant food-borne pathogenic bacteria.
9. Use of an active strain of a drug-resistant food-borne pathogenic bacterium-inhibiting agent according to claim 1 or 2 for the preparation of a food preservative.
10. A food preservative comprising the active strain of claim 1 or 2.
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