CN114917239A - Application of pomegranate polyphenol in prevention and control of vibrio parahaemolyticus - Google Patents
Application of pomegranate polyphenol in prevention and control of vibrio parahaemolyticus Download PDFInfo
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- CN114917239A CN114917239A CN202210411849.2A CN202210411849A CN114917239A CN 114917239 A CN114917239 A CN 114917239A CN 202210411849 A CN202210411849 A CN 202210411849A CN 114917239 A CN114917239 A CN 114917239A
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- vibrio parahaemolyticus
- punicalagin
- antibacterial agent
- food
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Classifications
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/90—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having two or more relevant hetero rings, condensed among themselves or with a common carbocyclic ring system
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention discloses an application of pomegranate polyphenol in inhibiting vibrio parahaemolyticus, belonging to the field of food-borne pathogenic microorganism control. The pomegranate polyphenol punicalagin has a killing effect on vibrio parahaemolyticus mainly by damaging cell membranes and influencing protein synthesis, and has little interference effect on bacterial genome DNA. Compared with the traditional antibacterial agent which acts on intracellular targets to kill microorganisms, the punicalagin acts on cell membranes or influences the protein metabolism of bacteria to inhibit the bacteria, can make pathogenic bacteria difficult to generate drug resistance, and has wide application value in the fields of food industry, aquaculture and the like which are easily polluted by vibrio parahaemolyticus.
Description
Technical Field
The invention relates to application of pomegranate polyphenol in vibrio parahaemolyticus prevention and control, and belongs to the technical field of pathogenic microorganism control.
Background
Vibrio parahaemolyticus (Vibrio parahaemolyticus) is a common food-borne pathogenic bacterium, widely distributed in offshore waters, estuaries and seabed sediments, and is one of the most severely polluted pathogenic bacteria in marine products. The natural bacteria carrying rate of the strain in aquatic products is high, the detection rate of the strain in warm seasons is 90%, the detection rate of the strain in warm seasons is 70%, the detection rate of the strain in warm seasons is 60%, the detection rate of the strain in crabs is 40%, and the average detection rate of the strain in aquatic products is 65%. Eating raw or uncooked aquatic products contaminated by the bacteria is very likely to cause gastroenteritis symptoms such as nausea, vomiting, diarrhea, abdominal pain, etc. Since 1950, the occurrence scale and population exposure of food poisoning caused by vibrio parahemolyticus have obviously increased, and the first microbial food poisoning is jumped over.
At present, in the food industry and the aquaculture industry, in order to kill aquatic product and water environment pollution caused by vibrio parahaemolyticus and clinically control organism infection caused by the vibrio parahaemolyticus, chemical bactericides, disinfectants or antibiotics are mainly used, large-scale use of the disinfectants and the antibiotics causes the vibrio parahaemolyticus in nature to generate drug resistance or even multiple drug resistance, and the drug-resistant vibrio parahaemolyticus can be spread in a food chain and can enter a human body along with the food chain through a horizontal gene transfer mechanism. In addition, the bacteria can form biofilms on various surfaces or interfaces (such as the surfaces of chopping boards, equipment and the like for food processing), and the resistance of biofilm cells to the disinfectant is about 1000 times that of planktonic cells. This presents a significant challenge to food safety and public health.
CN113262245A discloses the use of dogwood extract in the preparation of drugs for inhibiting marine pathogenic bacteria, but the MIC value of dogwood alcohol extract to Vibrio parahaemolyticus is 15.63mg/mL, and the dogwood alcohol extract needs to be used in large quantities in practical application to possibly exert antibacterial effect. CN112616862A discloses a composite plant extract for inhibiting vibrio parahaemolyticus in an aqueous environment, and a preparation method and application thereof, wherein the preparation process of an antibacterial composite is complicated, the antibacterial application is limited to be applied in the aqueous environment, and the application range is narrow.
Disclosure of Invention
[ problem ] to
The invention aims at solving the increasingly serious problems of vibrio parahaemolyticus pollution and bacterial drug resistance in the current marine food industry and aquaculture industry, and aims to provide the application of pomegranate polyphenol punicalagin in inhibiting vibrio parahaemolyticus.
[ solution ]
The invention provides application of pomegranate polyphenol-Punicalagin (CAS No.: 65995-63-3) in inhibiting growth of vibrio parahaemolyticus, controlling vibrio parahaemolyticus pollution, controlling infection of vibrio parahaemolyticus pollution and resisting vibrio parahaemolyticus biofilm formation. The vibrio parahaemolyticus includes a food poisoning isolate and a drug-resistant pandemic strain.
The inhibition of the growth of the vibrio parahaemolyticus refers to a process that the growth of the vibrio parahaemolyticus is delayed, slow or not grown or even killed compared with the growth of the vibrio parahaemolyticus which is not subjected to any treatment, namely the inhibition of the growth of the vibrio parahaemolyticus.
The control of the vibrio parahaemolyticus pollution refers to the process of inhibiting or weakening the growth, the field planting and the virulence production of the vibrio parahaemolyticus on any biological (such as shrimps, fishes, scallops and the like) or non-biological surface, liquid level and interface (such as aquatic product culture environment, processing tool surface and the like).
The control of the vibrio parahaemolyticus infection refers to the inhibition of the survival of the vibrio parahaemolyticus in the body and the weakening of the pathogenic process of the vibrio parahaemolyticus.
The punicalagin has effects of resisting Vibrio parahaemolyticus biofilm formation and resisting toxicity.
The invention provides a medicine, an antibacterial agent for food or an antibacterial agent for cultivation for treating interference of vibrio parahaemolyticus, which contains punicosides.
The antibacterial agent can contain any natural product which can perform antibacterial action with punicalagin besides punicalagin.
The dosage form of the antimicrobial agent may be a fluid, semi-fluid, or solid powder, and the like.
In the antibacterial agent, the effective content of punicalagin is more than or equal to the minimum inhibitory concentration meter.
The antimicrobial agent is used to inhibit the growth of Vibrio parahaemolyticus, thereby avoiding adverse consequences caused by contamination or contamination with Vibrio parahaemolyticus.
[ advantageous effects ]
The non-chemically synthesized, non-antibiotic and good-water-solubility natural polyphenol compound punicalagin can effectively inhibit the growth of vibrio parahaemolyticus and drug-resistant strains thereof, and is convenient for developing a method for preventing, controlling and controlling food-borne pathogenic bacteria in a green and safe manner.
The minimum bactericidal concentration of punicalagin to Vibrio parahaemolyticus is 200-300 mug/mL, and the minimum bacteriostatic concentration is 150-200 mug/mL. Low concentrations of punicalagin (3.125, 6.25 and 12.5. mu.g/mL) can inhibit Vibrio parahaemolyticus biofilm formation and virulence gene expression.
Punicalagin exerts a bactericidal effect by destroying the cell membrane permeability of vibrio parahaemolyticus, causing partial leakage of substances in cells, influencing the synthesis of bacterial protein, and causing the physiological metabolic function abnormality of vibrio parahaemolyticus.
Punicalagin has little interference effect on bacterial genome DNA. Compared with the traditional antibacterial agent which acts on intracellular targets to kill microorganisms, the antibacterial agent inhibits bacteria by acting on cell membranes or influencing bacterial protein metabolism, can make pathogenic bacteria difficult to generate drug resistance, and has wide application value in the fields of food industry, aquaculture and the like which are easily polluted by vibrio parahaemolyticus.
The result of in vivo anti-infection experiments shows that punicalagin can control the in vivo infection effect of vibrio parahaemolyticus to a certain extent, reduce the production of proinflammatory factors of mice infected by vibrio parahaemolyticus, increase the production of anti-inflammatory factors, reduce the vibrio parahaemolyticus loading amount, intestinal permeability and short-chain fatty acid yield in mouse feces, and reduce the death rate of mice. Therefore, the punicalagin has wide application value and reference significance in the inhibition effect and anti-infection effect on the vibrio parahaemolyticus in vivo and in vitro, in the fields of aquaculture, food industry and the like which are easily polluted by the vibrio parahaemolyticus or in the aspect of clinically controlling the vibrio parahaemolyticus infection.
Drawings
FIG. 1 shows the evaluation of the bactericidal effect of punicalagin with different concentrations on Vibrio parahaemolyticus in example 2 of the present invention;
FIG. 2 is a graph showing the effect of punicalagin at different concentrations on the metabolic activity of Vibrio parahaemolyticus in example 2 of the present invention;
FIG. 3 is a graph showing the effect of punicalagin of different concentrations on the permeability of parahemolytic vibrio membrane in example 2 of the present invention;
FIG. 4 is a scanning electron micrograph of Vibrio parahaemolyticus treated with punicalagin at different concentrations in example 2 of the present invention;
FIG. 5 shows the effect of punicalagin with different concentrations on the mycoprotein of Vibrio parahaemolyticus in example 2 of the present invention;
FIG. 6 is the effect of punicalagin at different concentrations on Vibrio parahaemolyticus genomic DNA according to example 2 of the present invention;
FIG. 7 shows the effect of punicalagin at different concentrations on the amount of Vibrio parahaemolyticus biofilm formed in example 3 of the invention;
FIG. 8 is a scanning electron micrograph of Vibrio parahaemolyticus biofilm treated with punicalagin at different concentrations in example 3 of the present invention;
FIG. 9 shows the effect of punicalagin with different concentrations on the expression level of virulence related genes of Vibrio parahaemolyticus in example 3 of the present invention;
FIG. 10 is a graph of the effect of punicalagin treatment on mortality of infected mice according to example 4 of the present invention;
FIG. 11 is a graph showing the effect of punicalagin treatment on Vp bacterial load of infected mice in example 4 of the present invention;
FIG. 12 is a graph of the effect of punicalagin treatment on intestinal permeability in infected mice according to example 4 of the present invention;
FIG. 13 is a graph of the effect of punicalagin treatment on short chain fatty acid production in infected mice according to example 4 of the present invention;
FIG. 14 is a graph of the effect of punicalagin treatment on the levels of inflammatory factors in infected mice according to example 4 of the present invention.
Detailed Description
The invention is further illustrated by the following specific examples. The examples are only for illustrating the present invention and are not intended to limit the scope of the present invention. The experimental methods described in the examples of the present invention are all conventional methods unless otherwise specified. The materials, reagents and the like used are commercially available unless otherwise specified.
Example 1: evaluation of antibacterial activity of punicalagin on Vibrio parahaemolyticus
S1 test strains
Vibrio parahaemolyticus ATCC17802, Vibrio parahaemolyticus ATCC 33847 and Vibrio parahaemolyticus RIMD2210633 (named th035) having streptomycin resistance were selected as experimental strains.
S2 activation of test strains
Inoculating the parahemolytic arc strain frozen at-80 deg.C to 10mL of 3% NaCl TSB (+ -200 μ g/mL Sm) broth, activating, streaking on 3% NaCl TSA (+ -200 μ g/mL Sm) plate, inoculating single colony in 30mL of TSB, culturing at 37 deg.C overnight in a shaking table to make the bacteria in late logarithmic growth stage, centrifuging at 5000 × g and 4 deg.C for 5min, removing supernatant, washing with PBS for 3 times, and adjusting the concentration of bacterial suspension to about 10 8 CFU/mL, spare.
S3 Minimum Inhibitory Concentration (MIC) determination
Dissolving punicalagin in sterile water to prepare mother liquor, filtering and sterilizing through a 0.22 mu m filter membrane, and adding the mother liquor into sterilized 3% NaCl TSB (+/-200 mu g/mL Sm) broth to prepare a series of punicalagin twofold dilutions. Taking the prepared twice diluted solution of punicalagin and the vibrio parahaemolyticus solution (10) diluted in equal volume 6 CFU/mL) to obtain a mixed solution, transferring the mixed solution to a 96-well cell culture plate, and adding 200 mu L of the mixed solution into each well to ensure that the final concentration gradient of the punicalagin is between 0 and 1 mg/mL. Each concentration is 3 parallels, a series of twice-diluted punicalagin solutions without adding bacterial liquid are used as background blank control, and vibrio parahaemolyticus solution without adding punicalagin is used as negative control. After standing and culturing for 24h at 37 ℃, observing the turbidity of the culture solution in each hole by naked eyes, wherein the lowest punicalagin concentration without vibrio parahaemolyticus growth (clarification in the hole) is the Minimum Inhibitory Concentration (MIC), further measuring the absorbance value at the wavelength of 600nm, and if the difference between the absorbance values before and after culturing is less than 0.05, determining that the test concentration can inhibit the growth of bacteria and determining the test concentration as the Minimum Inhibitory Concentration (MIC) of punicalagin.
S4 measurement of Minimum Bactericidal Concentration (MBC)
100. mu.L of the wells capable of inhibiting the growth of Vibrio hemolyticus in S3 were spread on a sterile 3% NaCl TSA plate, and cultured in an incubator at 37 ℃ for 48 hours. The minimum concentration at which the test microorganism was killed by 99.9% (the number of cells was reduced by 3log or more) as compared with the inoculation concentration was referred to as the Minimum Bactericidal Concentration (MBC), and the results are shown in Table 1.
TABLE 1 inhibition results of punicalagin on Vibrio parahaemolyticus
As shown in Table 1, punicalagin has good inhibiting and killing effects on Vibrio parahaemolyticus isolates of different sources, the Minimum Inhibitory Concentration (MIC) is 150-200 mug/mL, and the Minimum Bactericidal Concentration (MBC) is 200-300 mug/mL. According to the MIC value, the bactericidal effect target of punicalagin on Vibrio parahaemolyticus is further investigated.
Example 2: sterilization mechanism of punicalagin on vibrio parahaemolyticus
S1 sterilizing effect of punicalagin on vibrio parahaemolyticus
The influence of punicalagin on the bactericidal effect of vibrio parahaemolyticus is determined by a colony counting method. Specifically, punicalagin was added to fresh sterile 3% NaCl TSB (+ -200. mu.g/mL Sm) broth to final concentrations of 4 × MIC, 2 × MIC, 1 × MIC, and 0 (control), with three replicates per concentration set. Respectively inoculating 1% (v/v) of fresh bacteria suspension adjusted in advance to make final concentration of thallus be 2 × 10 5 CFU/mL, mixing well and shake culturing in a shaker at 37 ℃. The bacterial suspension is sucked in 2h, 4h and 8h respectively, after ten-fold dilution of series gradient, 100 mu L of the bacterial suspension is coated on a 3% NaCl TSA plate, and colony counting is carried out after 24h of culture.
Results as shown in figure 1, the colony concentration of the punicalagin treated group decreased significantly with increasing punicalagin treated concentration (1 × MIC, 2 × MIC, 4 × MIC) and longer exposure time (2h, 4h, and 8h) compared to the untreated group (i.e., control). Based on the good bactericidal effect of punicalagin on vibrio parahaemolyticus.
S2 influence of punicalagin on intracellular ATP of parahemolytic vibrio
The concentration of the washed fresh bacterial liquid is adjusted to be about 10 8 CFU/mL. 2mL of the bacterial suspension was added to the centrifuge tube, and the punicalagin solution was added to give final concentrations of 4 XMIC, 2 XMIC, 1 XMIC, and 0 (control), respectively, and the sample was incubated in an incubator at 37 ℃ for 1 h. Centrifuging the cultured bacterial solution at 5000 × g 4 deg.C for 5min, collecting bacterial cells, adding 1mL ATP extractive solution, ultrasonic crushing for 1min (ice bath, intensity of 200W, ultrasonic treatment for 10s, stopping 5s), and centrifuging at 10000 × g 4 deg.C for 5 min; collecting supernatant, adding 500 μ L chloroform, shaking, centrifuging at 10000 × g 4 deg.C for 3min, collecting supernatant, and placing on ice. Then, the ATP content in the parahemolytic vibrio cells is measured according to the instruction of the ATP content detection kit.
As shown in fig. 2, ATP is an energy substance for cell metabolism, and measurement of ATP content in the cell of the bacterium reflects the intracellular metabolic capacity of the bacterium, and compared with the untreated group (i.e., the control group), the intracellular ATP in the punicalagin-treated group was significantly reduced and exhibited a certain dose dependence. The results indicate that punicalagin may cause abnormal intracellular metabolism or altered cell membrane permeability of parachlorovibrio parahaemolyticus. The effect of punicalagin on the permeability of parahemolytic vibrio membranes was then further examined.
S3 influence of punicalagin on leakage of Vibrio parahaemolyticus ions
The concentration of the washed fresh bacterial liquid is adjusted to be about 10 8 CFU/mL. Taking 10mL of the bacterial liquid, adding punicalagin solution to make the final concentrations of the punicalagin solution be 4 × MIC, 2 × MIC, 1 × MIC and 0 (control group), setting a background blank control, and placing the sample in an incubator at 37 ℃ for culturing for 1 h. Centrifuging the cultured bacteria solution at 5000 Xg 4 deg.C for 5min, collecting supernatant, filtering with 0.22 μm filter membrane, and measuring potassium ion (K) in the filtrate with atomic absorption spectrophotometer + ) And KCl solutions with different concentrations are used as a standard curve (0-10 mg/L). Likewise, the amount of protein leakage in the supernatant was determined using the Bradford method; measurement Using Nanodrop ultramicro SpectrophotometerAmount of nucleic acid leakage in supernatant.
Intracellular ion/molecule leakage may reflect changes in cell membrane permeability. FIG. 3 shows the leakage of intracellular potassium ions, proteins and nucleic acids from Vibrio parahaemolyticus when punicalagin acts on Vibrio parahaemolyticus for 2 h. The results show that the potassium ion leakage and protein leakage of the punicalagin treated vibrio parahaemolyticus are remarkably increased and show certain concentration dependence compared with the untreated group (control group). Nucleic acid leakage, however, also increased significantly above 2 × MIC concentrations. This suggests that the cell membrane may be a target for punicalagin to exert bactericidal effects. Furthermore, the influence of punicalagin treatment on the cell morphology of the vibrio parahaemolyticus can be visually observed by adopting a scanning electron microscope.
S4 influence of punicalagin on cell morphology of Vibrio parahaemolyticus
The concentration of the washed fresh bacterial liquid is adjusted to be about 10 8 CFU/mL. Taking the above bacterial liquid, adding punicalagin solution to make final concentration 4 × MIC, 2 × MIC, 1 × MIC and 0 (control group), and culturing at 37 deg.C for 4-6 h. Centrifuging the cultured bacterial solution at 4 deg.C for 5min at 5000 Xg, removing supernatant, collecting thallus, and fixing with 2.5% glutaraldehyde solution at 4 deg.C for 4-6 h. The fixed sample is centrifuged for 5min at the temperature of 5000 Xg 4 ℃, the supernatant is discarded, the thalli are washed for 3 times by sterile physiological saline, and then the thalli are respectively eluted by 30 percent, 50 percent, 70 percent, 90 percent and 100 percent ethanol for 15min each time. Finally, the sample is dripped into a special sterile cover glass, and the morphology of the bacterial cells is observed by using a field emission scanning electron microscope.
As can be seen from FIG. 4, Vibrio parahaemolyticus which is not treated with punicalagin is rod-shaped, has a full shape, two ends are blunt, the surface of the cell body is smooth and flat, and is typically short rod-shaped. After 1 × MIC punicalagin treatment, shrinkage and collapse appear on part of the bacterial surface; after 2 × MIC punicalagin treatment, severe shrinkage occurred on the surface of the bacteria and a portion of the bacteria of ATCC17802 strain began to shed contents. After 4 XMIC punicalagin treatment, severe collapse and damage of the bacterial surface occur, and the result further verifies that punicalagin treatment can destroy the cell membrane of Vibrio parahaemolyticus.
S5 influence of punicalagin on mycoprotein and DNA of vibrio parahaemolyticus
Adopting a gram-negative bacteria protein extraction kit, and extracting mycoprotein of vibrio parahaemolyticus which is not treated and is treated by punicalagin (4 × MIC, 2 × MIC and 1 × MIC) with different concentrations according to the kit instruction. The BCA method was used to determine mycoprotein concentrations and the effect of punicalagin treatment on bacterial proteins was examined in conjunction with SDS-PAGE gel electrophoresis. Similarly, the bacterial genomic DNA extraction kit was used to extract the genomic DNA of Vibrio parahaemolyticus untreated and treated with punicalagin at different concentrations (4 × MIC, 2 × MIC, 1 × MIC) according to the kit instructions. 5.0. mu.L of the extracted genomic DNA was subjected to electrophoresis on 1% agarose gel to examine the integrity and purity of the DNA.
The SDS-PAGE gel electrophoresis result of figure 5 shows that along with the increase of the treatment concentration of the punicalagin, the brightness of most protein bands (45, 55, 75, 80, 95-150kDa) of the thalli gradually becomes lighter and even disappears, and the brightness of 1 protein band at the position of 35-42kDa increases, which indicates that the punicalagin has a certain inhibiting effect on the synthesis of partial proteins of bacteria.
FIG. 6 shows that the bacterial DNA mobility did not change during agarose gel electrophoresis, indicating that punicalagin did not or very little interact with DNA.
From the results of this example, it can be seen that the cell membrane is an important target for punicalagin to kill Vibrio parahaemolyticus. The punicalagin can cause partial leakage of substances in cells by damaging the permeability of cell membranes, and can influence the metabolism of bacterial proteins, finally cause the abnormal physiological metabolism function of vibrio parahaemolyticus, and further play a role in sterilization. It is worth mentioning that punicalagin has little interference on the vibrio parahaemolyticus genomic DNA. Research shows that compared with the traditional antibacterial agent which acts on intracellular targets to kill microorganisms, the antibacterial agent acts on cell membranes or influences the protein metabolism of bacteria to inhibit bacteria, so that pathogenic bacteria are difficult to generate drug resistance.
Example 3: inhibition effect of punicalagin on vibrio parahaemolyticus biofilm and virulence gene expression
S1 influence of punicalagin on Vibrio parahaemolyticus biofilm formation amount
Taking fresh bacterial liquid (10) cultured overnight after activation 9 CFU/mL) was transferred to 96-well cell culture plates at 250 μ L per well, punicalagin solution was added to make the final concentration of punicalagin 12.5, 6.25, 3.125 μ g/mL, respectively, and cultured at 37 ℃ for 24, 48 and 72h with no punicalagin added as a control. The concentration of the cultured suspension was measured at a wavelength of 630nm, the suspension was removed, and the suspension was washed with PBS 3 times to remove floating cells. The 96-well plate was then dried at 50 ℃ for 20min, 250 μ L of 1% crystal violet stain was added and stained at room temperature for 20min, the crystal violet stain in the plate was aspirated and washed 3 times with sterile water to remove unbound dye. And then the washed pore plate is placed at room temperature for natural drying for 30min, 250 mu L of 33% glacial acetic acid is added into each pore, and the pore plate is placed still for 20min and then the absorbance value at 570nm is measured. Biofilm formation amount OD 570nm /OD 630nm And (6) counting.
As shown in fig. 7, the punicalagins (3.125, 6.25 and 12.5 μ g/mL) at different concentrations significantly inhibited biofilm formation of vibrio parahaemolyticus ATCC17802 at 24h, 48h and 72h, and exhibited a concentration-dependent pattern, and the inhibition rates (inhibition rate (control biofilm formation amount-treatment biofilm formation amount)/control biofilm formation amount × 100%) were 20.9 to 51.5%, 24.5 to 52.4% and 28.9 to 65.5%, respectively, as compared to the untreated group (i.e., control group); similarly, punicalagin inhibits the biofilm formation of vibrio parahaemolyticus th035 more greatly in a concentration-dependent mode, and the inhibition rate is as high as 40.2-74.4%.
S2 influence of punicalagin on Vibrio parahaemolyticus biofilm morphology
Taking fresh bacterial liquid (10) cultured overnight after activation 9 CFU/mL) at 500. mu.L per well to pre-load sterile paddles
The 24-well cell culture plate was incubated at 37 ℃ for 48 hours with addition of punicalagin solutions (final concentrations of 0, 3.125, 6.25 and 12.5. mu.g/mL). The biofilm was washed 3 times gently and fixed with 2.5% glutaraldehyde solution for 4-6h at 4 ℃. Then the biomembrane sample is gradiented by 30 percent, 50 percent, 70 percent, 90 percent and 100 percent ethanol respectivelyAnd (5) eluting for 15min each time. The biofilm samples were dried overnight and the biofilm formation status was visually observed using a field emission scanning electron microscope.
As is apparent from fig. 8, punicalagin inhibited vibrio parahemolyticus from adhering to the slide and prevented the formation of a biofilm, compared to the untreated group (i.e., the control group).
S3 influence of punicalagin on Vibrio parahaemolyticus virulence gene expression
Extracting the vibrio parahaemolyticus total RNA which is not treated and is treated by punicalagin (3.125, 6.25 and 12.5 mu g/mL) with different concentrations by adopting a bacterial total RNA extraction kit according to the kit instruction. The Nanodrop measures the mass concentration of the extracted RNA, and adjusts the RNA concentration of the qualified RNA to be uniform. Total RNA was reverse transcribed into cDNA using a reverse transcription kit. Then, 16s RNA is taken as an internal reference gene, functional genes related to the formation and the toxicity of the vibrio parahaemolyticus biofilm are selected as target amplification genes to carry out qRT-PCR reaction, and 2 is adopted -ΔΔCt The method evaluates the relative expression changes of the target gene.
From fig. 9, 6.25, 12.5 μ g/mL punicalagin significantly (p <0.01) down-regulated the expression levels of vibrio parahaemolyticus biofilm and virulence related genes (ompW, vp0950, vp0952, vopQ, vpA0450, flgL, luxS, aphA).
Example 4: effect of punicalagin on prevention of Vibrio parahaemolyticus infection
S1 influence of punicalagin on mortality and Vp bacterial load of infected mice
SPF grade 4-week-old C57BL/6J male mice (15 + -1 g) were selected and randomly divided into 2 groups, namely punicalagin treatment group (Pu + Vp) and infection group (Vp). Mice were acclimatized for 1 week, gavaged for 4 weeks, weekly for body mass and observed for status. Gavage the mice about 10 9 Infecting with CFU Vibrio parahaemolyticus liquid, observing mental state and death condition of mice within 1 week after infection, collecting feces of mice 12, 24, 36 and 48h after infection, and counting Vp capacity in feces of infected mice.
It is known from fig. 10 that punicalagin treatment can reduce the mortality rate of vibrio parahemolyticus infected mice to some extent, and the death of mice generally occurs within 2 days after infection. Further, we counted the Vibrio parahaemolyticus load in the feces of mice within 2 days after infection.
As seen in fig. 11, punicalagin treatment reduced vibrio parahemolyticus (Vp) load in the feces of mice 12h and 24h post infection.
S2 influence of punicalagin on intestinal permeability of infected mice
Mice were fasted for 4h at approximately 20h post-infection, and then gavaged with 100 μ L fluorescein isothiocyanate-dextran (FITC-dextran, 80mg/mL, 4000 Da). The mice are sacrificed after 4h of gastric lavage, the blood of the mice is taken, the fluorescence intensity of the blood of each group of mice is measured by a fluorescence microplate reader, and the excitation wavelength and the emission wavelength are 485 nm and 535nm respectively. Meanwhile, a standard curve is prepared by using fluorescein isothiocyanate-dextran double gradient diluent.
Intestinal permeability is the most important index reflecting mechanical barrier, once food, bacteria and other substances enter the circulatory system through the intestinal barrier, the immune system recognizes the substances as foreign matters, so that the body and the intestinal tract are inflamed, and the permeability is further increased. It is seen from figure 12 that punicalagin treatment very significantly reduced intestinal permeability in infected mice.
S3 influence of punicalagin on short-chain fatty acids in infected mice
The mouse feces of each group were taken into a 2mL centrifuge tube, the glass residue was added, diluted sulfuric acid was added for acidification (50%, v/v), ether and internal standard were added, and vortex oscillation was performed for 3 minutes twice. Ultrasonic treating in ice water bath for 30min, standing at-20 deg.C for 30min, and centrifuging at 12000g and 4 deg.C for 15 min. The supernatant was transferred to a 1.5mL centrifuge tube. Adding 250mg anhydrous sodium sulfate, vortexing, centrifuging at 12000g 4 deg.C for 15min, collecting supernatant, filtering with 0.22 μm organic filter membrane, and performing on-machine analysis with gas chromatography-mass spectrometer. Chromatographic conditions are as follows: the temperature of a sample inlet is 260 ℃; the sample volume is 1 mu L; the split ratio is 50: 1; the solvent delay was 2.5 min. Temperature rising procedure: an initial temperature of 80 ℃; the temperature is programmed to 120 ℃ at 40 ℃/min, the temperature is raised to 200 ℃ at 10 ℃/min, and the temperature is kept for 2 min. Then the operation is carried out at 230 ℃ for 3 min. Mass spectrum conditions: the electron bombardment ion source (EI) has the ion source temperature of 230 ℃, the quadrupole rod temperature of 150 ℃, the transmission line temperature of 250 ℃ and the electron energy of 70 eV. The scanning mode is a full SCAN mode (SCAN), and the mass scanning range is as follows: m/z: 30-300.
Short chain fatty acids SCFAs exert a variety of effects locally. For example, SCFAs are used in anaerobic environments to maintain intestinal redox balance; SCFAs can maintain the integrity of the intestinal wall barrier and prevent intestinal inflammation. The intestinal wall barrier is a physical barrier formed by epithelial cells through intercellular junctions, which promotes nutrient absorption and prevents the passage of harmful substances and pathogens into the intestinal tract through the extracellular pathways. It is known from figure 13 that punicalagin treatment can reduce short chain fatty acid production in infected mice to some extent.
S4 influence of punicalagin on inflammatory factors of infected mice
After the mice are killed, blood is taken from eyeballs, the blood is placed at room temperature for 30-60min, centrifuged at 2000 Xg 4 ℃ for 20min, serum is sucked, and split-packaged and stored at-80 ℃. Then, the levels of inflammatory factors IL-6, IL-beta, IL-10, TNF-alpha and IFN-gamma in the serum of each group of mice are detected by adopting an ELISA kit according to the requirements of the instruction.
When tissues are damaged or infected by toxins or bacteria, an inflammatory response of the body is induced, and the damaged cells are induced to release inflammatory factors. Wherein, IL-1 beta, IL-6, TNF-alpha and IFN-gamma are typical proinflammatory cytokines and participate in the up-regulation of inflammatory response; IL-10 is a typical anti-inflammatory factor and plays an important role in the homeostasis of immune responses. As shown in FIG. 14, punicalagin treatment significantly reduced the levels of proinflammatory factors such as IL-1 beta, IL-6, TNF-alpha and IFN-gamma in infected mice; increasing the level of IL-10 anti-inflammatory factor.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by one skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (10)
1. Application of punicosides in preparing products for inhibiting growth of Vibrio parahaemolyticus, controlling contamination of Vibrio parahaemolyticus, controlling infection of Vibrio parahaemolyticus or resisting biofilm formation of Vibrio parahaemolyticus is provided.
2. Use according to claim 1, characterized in that said punicosides are punicalagins.
3. The use of claim 1, wherein the vibrio parahaemolyticus comprises a food poisoning isolate and a drug resistant pandemic strain.
4. Use according to claim 1, characterized in that the product comprises: can be used for treating interference of Vibrio parahaemolyticus, food antibacterial agent, and breeding antibacterial agent.
5. The use according to claim 1, wherein the food-grade antimicrobial agent and the farming antimicrobial agent are bacterial antimicrobial agents.
6. A pharmaceutical, food-grade or aquaculture antimicrobial agent for treating interference of Vibrio parahaemolyticus, characterized by containing punicosides.
7. The drug, the antibacterial agent for food or the antibacterial agent for aquaculture for treating interference of vibrio parahemolyticus according to claim 6, wherein said punicosides are punicalagin.
8. The drug, the antibacterial agent for food or the antibacterial agent for aquaculture for treating interference of Vibrio parahaemolyticus according to claim 6, wherein the drug contains any substance that can synergistically exert bacteriostatic action with punicalagin.
9. The drug, the antibacterial agent for food or the antibacterial agent for aquaculture for use in the treatment of interference of Vibrio parahaemolyticus according to claim 6, wherein the dosage form of the antibacterial agent is fluid, semi-fluid or solid powder.
10. The drug, the antibacterial agent for food or the antibacterial agent for culture for treating interference of Vibrio parahaemolyticus according to claim 6, wherein the effective content of punicalagin is greater than or equal to the minimum inhibitory concentration.
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