CN106306979A - Application of spice extract to inhibition and removal of food-borne pathogenic bacterium biological membrane - Google Patents
Application of spice extract to inhibition and removal of food-borne pathogenic bacterium biological membrane Download PDFInfo
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- CN106306979A CN106306979A CN201610687579.2A CN201610687579A CN106306979A CN 106306979 A CN106306979 A CN 106306979A CN 201610687579 A CN201610687579 A CN 201610687579A CN 106306979 A CN106306979 A CN 106306979A
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- 239000000284 extract Substances 0.000 title claims abstract description 100
- 235000013599 spices Nutrition 0.000 title claims abstract description 39
- 230000005764 inhibitory process Effects 0.000 title abstract description 7
- 239000012528 membrane Substances 0.000 title abstract description 7
- 244000052616 bacterial pathogen Species 0.000 title abstract description 6
- 238000000034 method Methods 0.000 claims abstract description 15
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 14
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 9
- 150000004676 glycans Chemical class 0.000 claims description 34
- 229920001282 polysaccharide Polymers 0.000 claims description 34
- 239000005017 polysaccharide Substances 0.000 claims description 34
- 235000011347 Moringa oleifera Nutrition 0.000 claims description 33
- 241000220215 Moringa Species 0.000 claims description 27
- 241000607598 Vibrio Species 0.000 claims description 26
- 241000191967 Staphylococcus aureus Species 0.000 claims description 25
- 230000001629 suppression Effects 0.000 claims description 21
- 244000078673 foodborn pathogen Species 0.000 claims description 20
- 230000002401 inhibitory effect Effects 0.000 claims description 20
- 230000015572 biosynthetic process Effects 0.000 claims description 13
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- 238000002390 rotary evaporation Methods 0.000 claims description 3
- 238000012360 testing method Methods 0.000 claims description 3
- 241000305071 Enterobacterales Species 0.000 claims description 2
- 239000002994 raw material Substances 0.000 claims description 2
- 239000002893 slag Substances 0.000 claims description 2
- 239000012467 final product Substances 0.000 claims 1
- 238000002360 preparation method Methods 0.000 abstract description 3
- 238000011160 research Methods 0.000 abstract description 2
- 238000005260 corrosion Methods 0.000 abstract 1
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- 238000002481 ethanol extraction Methods 0.000 description 21
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- 230000002000 scavenging effect Effects 0.000 description 14
- 230000000844 anti-bacterial effect Effects 0.000 description 11
- 150000001875 compounds Chemical class 0.000 description 9
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- 244000179886 Moringa oleifera Species 0.000 description 6
- 238000013461 design Methods 0.000 description 6
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- FFRBMBIXVSCUFS-UHFFFAOYSA-N 2,4-dinitro-1-naphthol Chemical compound C1=CC=C2C(O)=C([N+]([O-])=O)C=C([N+]([O-])=O)C2=C1 FFRBMBIXVSCUFS-UHFFFAOYSA-N 0.000 description 2
- 206010018910 Haemolysis Diseases 0.000 description 2
- MJVAVZPDRWSRRC-UHFFFAOYSA-N Menadione Chemical compound C1=CC=C2C(=O)C(C)=CC(=O)C2=C1 MJVAVZPDRWSRRC-UHFFFAOYSA-N 0.000 description 2
- 206010048723 Multiple-drug resistance Diseases 0.000 description 2
- 230000008859 change Effects 0.000 description 2
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- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000008588 hemolysis Effects 0.000 description 2
- 230000002147 killing effect Effects 0.000 description 2
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- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
- 241001478240 Coccus Species 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 240000001131 Nostoc commune Species 0.000 description 1
- 235000013817 Nostoc commune Nutrition 0.000 description 1
- 241000896292 Odontothrips loti Species 0.000 description 1
- 235000019082 Osmanthus Nutrition 0.000 description 1
- 241000333181 Osmanthus Species 0.000 description 1
- 208000037581 Persistent Infection Diseases 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 108010059712 Pronase Proteins 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 241000209140 Triticum Species 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 241000607272 Vibrio parahaemolyticus Species 0.000 description 1
- 235000009392 Vitis Nutrition 0.000 description 1
- 241000219095 Vitis Species 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 235000011054 acetic acid Nutrition 0.000 description 1
- 150000001243 acetic acids Chemical class 0.000 description 1
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- 239000008280 blood Substances 0.000 description 1
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- 239000003814 drug Substances 0.000 description 1
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- 244000144992 flock Species 0.000 description 1
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- 235000013355 food flavoring agent Nutrition 0.000 description 1
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Classifications
-
- 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
- A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
- A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
- A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
- A23L3/3463—Organic compounds; Microorganisms; Enzymes
- A23L3/3472—Compounds of undetermined constitution obtained from animals or plants
-
- 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
Abstract
The invention provides application of a spice extract to inhibition and removal of a food-borne pathogenic bacterium biological membrane. The spice extract is a spice alcohol extract or a spice water extract. The biological membrane has high stress resistance, so the biological membrane can be controlled by combination of various methods. The invention provides theoretical basis for the application of the spice extract to control of the food-borne pathogenic bacterium biological membrane, and provides theoretical basis for research of a healthy and safe anti-corrosion anti-biological membrane preparation product.
Description
Technical field
The present invention relates to food safety and health field, particularly to spice extract at food-borne pathogens biomembrane
Suppression and the application removed.
Background technology
Food is all easily subject to the pollution of food-borne pathogens at the links of processing and storage and transport, in order to keep food
The fresh health of product, it is to avoid rot, people need to add various preservative in food, and wherein main with people
The chemical classes preservative of work synthesis is in the majority, but some chemical preservatives have certain hazardness to the mankind, to food seat belt
Carry out potential risks.
Biomembrane is that the Extracellular polymers (extracellular self produced is inlayed and is grown in microorganism
Polymeric substances, EPS, mainly extracellular polysaccharide, protein and extracellular dna) in micropopulation, how attached it is
The surface at object.Biomembrane is the microorganism a kind of adaptation to environment, is a kind of growth conditions corresponding with free state,
It can strengthen the drug resistance of antibacterial, environmental suitability and the toleration to host immune system attacks, and this is food safety and doctor
Serious risk is brought in treatment field.
Staphylococcus aureus (Staphylococcus aureus), escherichia coli (Escherichia coli) and pair
Hemolysis vibrion (Vibrio parahaemolyticus) is common food-borne pathogens, often causes food-safety problem, to people
Body health threatens, serious caused death.These pathogenic bacterium, in order to adapt to external environment, often flock together, and are formed
Biomembrane, thus resist the removing of the antibiotic substance such as external environment especially antibiotic, become the source of persistent infection.Tradition
Antibiotic substance poor to biomembranous action effect, and the method that present method is concentrated mainly on chemistry or biology, as changed
Learn the method such as synthetic drug, enzymolysis, these method complex process otherwise relatively costly, even human body is also had side effect.
Therefore develop novel, efficient, safe natural antibiont membrane substance and there is important theory and realistic meaning.Spice is for I
Flavoring agent common in daily life, and great majority all have the effect of bacteria growing inhibiting, but biomembrane is by it
No have suppression and scavenging action still to rarely have report.
Summary of the invention
In view of this, the technical problem to be solved, it is simply that propose spice extract at food-borne pathogens
Biomembrane suppression and the application removed.
For solving above-mentioned technical problem, the present invention is achieved by the following technical solutions:
Spice extract application in the suppression of food-borne pathogens biomembrane and removing, described spice extract is
Spice alcohol extraction thing or spice water extract.
Preferably, the one during described spice extract is the extract of Bulbus Allii, Cortex Cinnamomi, leaf of Moringa, Moringa stem, Flos Caryophylli
Or two kinds.
Preferably, described food-borne pathogens is staphylococcus aureus or escherichia coli or vibrio parahaemolytious.
Preferably, described spice extract is prepared as follows: spice obtains material slag for raw material break process, presses
Solid-liquid ratio 1:6～12 (m/v) adds extracting solution, with ultrasonic assistant extract, temperature is 40～60 DEG C, extraction time be 0.5～
1.5h, concentrates with Rotary Evaporators after decompression sucking filtration, and rotary evaporation condition is 50～60 DEG C, after proceeding to tool plug test tube constant volume
Obtain.
Preferably, described extracting solution is ethanol or the water of 80%.
Preferably, the suppression biomembranous to food-borne pathogens of described spice extract is embodied as suppressing food-borne
The biomembranous formation of pathogenic bacterium, suppression metabolic activity and suppression produce extracellular polysaccharide, and spice extract is to food-borne pathogenic
The minimal inhibitory concentration of bacterium is 6.25mg/mL-12.5mg/mL.Specifically, Moringa stem extract and Bulbus Allii extract are to golden yellow
Staphylococcic minimal inhibitory concentration is 12.5mg/mL；Colibacillary minimal inhibitory concentration is by Cortex Cinnamomi extract
6.25mg/mL, moringa oleifera leaf extractive is 12.5mg/mL to colibacillary minimal inhibitory concentration；Cortex Cinnamomi extract and Bulbus Allii carry
Take thing and the minimal inhibitory concentration of vibrio parahaemolytious is 6.25mg/mL.
Preferably, the removing biomembranous to food-borne pathogens of described spice extract is embodied as entering maturation
In biomembrane and kill wherein part food-borne pathogens and the extracellular polysaccharide of decomposing organism film.
Because biomembrane has stronger resistance, therefore multiple method should be used to combine biomembranous control, this
The bright application for spice extract in food-borne pathogens biomembrane controls provides theoretical basis, peace healthy for research
Full antibiont film product provides theoretical foundation.
Accompanying drawing explanation
Fig. 1 is the different spice extract big logotypes to staphylococcus aureus inhibition zone；
Fig. 2 is the different spice extract big logotypes to escherichia coli inhibition zone；
Fig. 3 is the different spice extract big logotypes to vibrio parahaemolytious inhibition zone；
Fig. 4 is that the compound of Moringa stem or the extract of Bulbus Allii and Moringa stem and Bulbus Allii extract is to Staphylococcus aureus
The MIC comparison diagram of bacterium；
Fig. 5 be Cortex Cinnamomi or the extract of leaf of Moringa and Cortex Cinnamomi with the compound of moringa oleifera leaf extractive to colibacillary
MIC comparison diagram；
Fig. 6 is the compound of Cortex Cinnamomi or the extract of Bulbus Allii and Cortex Cinnamomi and the Bulbus Allii extract MIC to vibrio parahaemolytious
Comparison diagram；
Fig. 7 is that the compound of Moringa stem or the extract of Bulbus Allii and Moringa stem and Bulbus Allii extract is to Staphylococcus aureus
The inhibition comparison diagram of bacterium biofilm formation；
Fig. 8 is Moringa stem and Bulbus Allii extract affects comparison diagram to Staphylococcus Aureus Biofilm metabolic activity；
Fig. 9 is Moringa stem and Bulbus Allii extract affects schematic diagram to staphylococcus aureus extracellular polysaccharide；
Figure 10 is that the compound of Cortex Cinnamomi or the extract of leaf of Moringa and Cortex Cinnamomi and moringa oleifera leaf extractive is raw to escherichia coli
Thing film formation amount affect comparison diagram；
Figure 11 is Cortex Cinnamomi and moringa oleifera leaf extractive affects comparison diagram to Escherichia coli biofilm metabolic activity；
Figure 12 is Cortex Cinnamomi and Moringa stem extract affects schematic diagram to escherichia coli extracellular polysaccharide；
Figure 13 is that the compound of Cortex Cinnamomi or the extract of Bulbus Allii and Cortex Cinnamomi and Bulbus Allii extract is biological to vibrio parahaemolytious
Film formation amount affect comparison diagram；
Figure 14 is Cortex Cinnamomi and Bulbus Allii extract affects comparison diagram to vibrio parahaemolytious biomembrane metabolic activity；
Figure 15 is Cortex Cinnamomi and Bulbus Allii extract affects schematic diagram to vibrio parahaemolytious extracellular polysaccharide；
Figure 16 is that Moringa stem extract, Bulbus Allii extract and their extract complex are raw to staphylococcus aureus
The clearance rate comparison diagram of thing film；
Figure 17 is that the Moringa stem of variable concentrations and Bulbus Allii extract are to staphylococcus aureus biofilm extracellular polysaccharide
Affect comparison diagram；
Figure 18 is that Cortex Cinnamomi extract, moringa oleifera leaf extractive and their extract complex are to Escherichia coli biofilm
Clearance rate comparison diagram；
Figure 19 is Cortex Cinnamomi and the moringa oleifera leaf extractive impact on E. coli mature biomembrane extracellular polysaccharide of variable concentrations
Comparison diagram；
Figure 20 is that Cortex Cinnamomi extract, Bulbus Allii extract and their extract complex are biomembranous to vibrio parahaemolytious
Clearance rate comparison diagram；
Figure 21 is Cortex Cinnamomi and the Bulbus Allii extract impact on vibrio parahaemolytious biofilm extracellular polysaccharide of variable concentrations
Comparison diagram；
Figure 22 is that CLSM observes the Bulbus Allii extract inhibitory action design sketch to Staphylococcus Aureus Biofilm；
Figure 23 is that CLSM observes the Cortex Cinnamomi extract inhibitory action design sketch to Escherichia coli biofilm；
Figure 24 is that CLSM observes Cortex Cinnamomi extract inhibitory action biomembranous to vibrio parahaemolytious design sketch；
Figure 25 is that CLSM observes the Bulbus Allii extract scavenging action design sketch to Staphylococcus Aureus Biofilm；
Figure 26 is that CLSM observes the Cortex Cinnamomi extract scavenging action design sketch to Escherichia coli biofilm；
Figure 27 is that CLSM observes Cortex Cinnamomi extract scavenging action biomembranous to vibrio parahaemolytious design sketch.
Detailed description of the invention
The present invention is got information about, below in conjunction with accompanying drawing, to the present invention for allowing those skilled in the art become apparent from
Preferred embodiment be described in detail.The experimental technique of unreceipted actual conditions in embodiment, generally according to normal condition
Or according to the condition proposed by manufacturer.
The preparation of the plant extracts such as embodiment 1 spice
By Fructus Foeniculi, Pericarpium Zanthoxyli, Fructus Piperis, Herba Rosmarini Officinalis, Cortex Cinnamomi, Flos Caryophylli be dried, pulverize, Bulbus Allii Cepae, leaf of Moringa, Moringa stem, Bulbus Allii,
Rhizoma Zingiberis Recens rubs with juice extractor respectively, is separately added into 80% ethanol (v/v) or deionized water by solid-liquid ratio 1:6～12 (m/v), then
Extracting with ultrasonic assistant, temperature is 40～60 DEG C, and extraction time is 0.5～1.5h.Enter with Rotary Evaporators after decompression sucking filtration
Row concentrates, and rotary evaporation condition is 50～60 DEG C, proceeds in tool plug test tube, and making mass concentration after constant volume is that (i.e. 1g perfume (or spice) is pungent for 1g/mL
Material extract constant volume is 1mL), it is stored in 4 DEG C of refrigerators standby after sealing.
The preparation of embodiment 2 bacteria suspension
Staphylococcus aureus, escherichia coli and vibrio parahaemolytious are inoculated in TSB culture medium respectively, are placed in shaking table
37 DEG C, 120r/min cultivates 12h.Thalline is collected, with 10mL 0.5 wheat prepared after 10 times of gradient dilutions by centrifugal for bacteria suspension
Family name opacity tube (0.25%BaCl20.2mL, 1%H2SO49.8mL) carry out turbidity to compare, and verify with colony counting method, finally
Determine that its viable bacteria content is about 108CFU/mL。
Embodiment 3 Odontothrips loti measures the bacteriostasis of spice extract
The TSA solid medium prepared adds 200 μ L and is diluted to 108The bacterium solution of CFU/mL, after coating uniformly, uses
Aseptic nipper is by sterilizing and is cooled to that the Oxford cup of room temperature is triangular in shape to be placed on flat board, adds 100 μ in the cup of each Oxford
The each extracting solution of L, observes after being placed in 37 DEG C of constant incubators cultivation 24h and measures antibacterial circle diameter, and experiment is with 80% second every time
Alcohol and sterilized water compare.The size of the inhibition zone of three kinds of food-borne pathogens is shown in accompanying drawing 1-3 by different extracts.
Embodiment 4 doubling dilution measures the spice extract minimal inhibitory concentration (MIC) to three kinds of bacterium
In 96 porocyte culture plates, it is initially charged the MH broth bouillon after 100 μ L sterilizings, then adds dilute at first row
Extract 100 μ L after releasing, therefrom draws 100 μ L in next column, sequentially into six Concentraton gradient, the most again after mix homogeneously
Adding 100 μ L concentration in every hole is 106The bacterium solution of CFU/mL, the concentration of such each column be respectively 50mg/mL, 25mg/mL,
12.5mg/mL, 6.25mg/mL, 3.13mg/mL and 1.56mg/mL.Each column three repetition, the 7th behavior is not added with extract and bacterium
The blank of liquid, the 8th behavior is not added with the negative control of extract.Minimum to three kinds of food-borne pathogens of different extracts
Mlc is shown in accompanying drawing 4-6, and " * * " represents that it is the most notable with matched group difference.
The quantitative determination spice extract impact on biofilm formation of embodiment 5 crystal violet method
In 96 orifice plates, add TSB culture medium, add extract and bacterium solution；Set blank and negative control simultaneously.
Close the lid, and use preservative film environmental sealing, put into the cultivation regular hour in constant incubator；Take out 96 orifice plates, discard training
Nutrient solution, and in every hole, add 250 μ L normal saline cleaning twice, remove flcating germ；96 orifice plates are put into 60 DEG C of forced air dryings
Machine is dried 15min；Adding the crystal violet solution of 200 μ L0.1% in every hole, dye 10min；Discard crystal violet solution,
The normal saline adding 250 μ L in every hole cleans three times, removes undyed crystal violet；60 DEG C of blast driers are dried
15min；In every hole, add 200 μ L33% glacial acetic acids, stand 10min.Its OD under 595nm wavelength is surveyed in microplate reader
Value.
Calculating suppression ratio:
Suppression ratio (%)=(matched group OD value-experimental group OD value)/matched group OD value × 100%.
Embodiment 6 XTT lowers method and surveys the metabolic activity in biomembrane
(1) in 96 orifice plates, TSB culture medium, extract and bacterium solution are added；Set blank and negative control simultaneously.Cover
Lid, uses preservative film environmental sealing, puts into the cultivation regular hour in constant incubator；
(2) take out 96 porocyte culture plates, discard content；
(3) in every hole, add 100 μ L PBS, then XTT solution and menadione solution are pressed 12.5:1 mixing
After, every hole adds 13.5 μ L, and lucifuge hatches 4h, and temperature is identical with cultivation temperature；
(4) under 420nm wavelength, its light absorption value is surveyed.
(5) suppression ratio is calculated
Suppression ratio (%)=(matched group OD value-experimental group OD value)/matched group OD value × 100%.
Embodiment 7 mensuration carries spice and takes the impact that extracellular polysaccharide is produced by thing
(1) bacterium solution after cultivating in the culture medium containing variable concentrations extract is centrifuged 10min in 5000r/min,
Remove supernatant, bacterium is resuspended in 10mL sterile saline, boils 10min, under room temperature, cool down 20min；
(2) adding the Pronase E of 50 μ L, 37 DEG C of lucifuges hatch 2h；
(3) adding the TCA solution that mass concentration is 10% of 200 μ L, 30min placed by frozen water；
(4) 10000r/min is centrifuged 30min, collects supernatant, adds isopyknic ethanol solution, places 1h for-20 DEG C；
(5) 10000r/min is centrifuged 20min, removes supernatant, precipitate with 95% washing with alcohol twice；
(6) final precipitation is resuspended in 1mL sterile deionized water, adds re-distilled phenol and the concentrated sulphuric acid of mass concentration 5%, boiling
Water-bath 10min；
(7) each sample is drawn 200 μ L and is added 96 porocyte culture plates, measures light absorption value under 420nm wavelength；
(8) suppression ratio is calculated
Suppression ratio (%)=(matched group OD value-experimental group OD value)/matched group OD value × 100%.
Embodiment 8 spice extract is on the impact of viable count in biofilm
In 50mL centrifuge tube, put into the coverslip after one piece of sterilizing, be subsequently adding 10mL TSB culture medium, access corresponding
Pathogenic bacterium so that it is final concentration is about 106CFU/mL.Staphylococcus aureus and escherichia coli are at 37 DEG C, and secondary haemolysis arc
Bacterium cultivates 48h at 30 DEG C.Coverslip taking-up normal saline after cultivating wash put into after three times new containing the denseest
The 50mL centrifuge tube of the 10mL normal saline of degree extract continues to cultivate 24h.Then coverslip is taken out, wash with normal saline
Put in the new centrifuge tube containing 10mL normal saline after three times, ultrasonic wave concussion 10min.After gradient dilution, take 100 μ L bacterium
Liquid is spread evenly across in TSA flat board, and relevant temperature observes counting after cultivating 24h.
Embodiment 9 laser confocal scanning microscope (CLSM) observes biomembranous stereochemical structure
(1) in Nostoc commune Vanch ware, put into the microscope slide of sterilizing, be subsequently adding 15mL and contain the TSB of variable concentrations extract
Culture medium, accesses strain, makes the final concentration in each culture dish reach 106CFU/mL；Staphylococcus aureus and escherichia coli
It is positioned over 37 DEG C, and vibrio parahaemolytious is placed in 30 DEG C of constant incubators and cultivates 24h；
(2) from culture dish, take out microscope slide, rinse three times with PBS, wipe surplus liquid with filter paper gently；
(3) on microscope slide, drip 5 μ L ConA-FITC mixed solutions under light protected environment, place 30min for 4 DEG C；
(4) in same position, drip 5 μ L PI solution again, place 15min for 4 DEG C；
(5) it is positioned in CLSM and observes biomembranous structure.
Embodiment 10 data statistics and analysis
Application SPSS22.0 software is averaged the calculating of value and standard deviation, and it is carried out significance analysis (P ＜
0.01 is that difference is extremely notable, and 0.01 ＜ P ＜ 0.05 is significant difference).
Embodiment 11 Bulbus Allii ethanol extraction, the suppression to Staphylococcus Aureus Biofilm of the Moringa stem ethanol extraction
Effect
After compounding to the Moringa stem of variable concentrations and the ethanol extraction of Bulbus Allii and their 1:1 (v/v), to golden yellow Fructus Vitis viniferae
The biomembranous formation of coccus suppresses.It was found that the formation of Staphylococcus Aureus Biofilm is had by these extracts
Inhibitory action, all shows stronger concentration dependant (accompanying drawing 7)；Outside to the metabolic activity of Staphylococcus Aureus Biofilm, born of the same parents
The generation of polysaccharide also has the strongest inhibitory action (accompanying drawing 8, accompanying drawing 9).Note: in figure, the concentration of extract is 1/ from low to high
8×MIC-2×MIC；Same letter represents that difference is not notable (P ＞ 0.05) between any two, and different letter representations are the poorest
Different notable (P ＜ 0.05)
Embodiment 12 leaf of Moringa ethanol extraction and the Cortex Cinnamomi ethanol extraction inhibitory action to Escherichia coli biofilm
After compounding to the leaf of Moringa ethanol extraction of variable concentrations and Cortex Cinnamomi ethanol extraction and their 1:1 (v/v), to greatly
The biomembranous formation of enterobacteria suppresses.It was found that these extracts have suppression to the formation of Escherichia coli biofilm
Effect, all shows stronger concentration dependant (accompanying drawing 10)；Metabolic activity, the generation of extracellular polysaccharide to Escherichia coli biofilm
Also there is the strongest inhibitory action (accompanying drawing 11, accompanying drawing 12).Note: in figure, the concentration of extract is 1/8 × MIC-2 from low to high
×MIC；Same letter represents that difference is not notable (P ＞ 0.05) between any two, different letter representation significant difference (P between any two
＜ 0.05)
Embodiment 13 Cortex Cinnamomi ethanol extraction and Bulbus Allii ethanol extraction inhibitory action biomembranous to vibrio parahaemolytious
After compounding to the Cortex Cinnamomi ethanol extraction of variable concentrations and Bulbus Allii ethanol extraction and their 1:1 (v/v), molten to pair
The biomembranous formation of blood vibrio suppresses.It was found that the formation biomembranous to big vibrio parahaemolytious of these extracts has
Inhibitory action, all shows stronger concentration dependant (accompanying drawing 13)；Metabolic activity biomembranous to vibrio parahaemolytious, extracellular polysaccharide
Generation also there is the strongest inhibitory action (accompanying drawing 14, accompanying drawing 15).
Embodiment 14 Bulbus Allii ethanol extraction, Moringa stem ethanol extraction are to staphylococcus aureus biofilm
Scavenging action
Staphylococcus aureus forms the biomembrane of maturation after cultivating 48h, by Moringa stem and the second of Bulbus Allii of variable concentrations
Alcohol extraction thing and their 1:1 (v/v) compound are added thereto, and set negative control simultaneously, continue to cultivate 24h；Survey the most respectively
Biofilm biomass, the generation amount of extracellular polysaccharide, calculate clearance rate.Result shows, these extracts are biological to staphylococcus aureus
Film has scavenging action (accompanying drawing 16)；Staphylococcus aureus extracellular polysaccharide is also had the strongest Scavenging activity (accompanying drawing 17).
Note: same letter represents that difference is not notable (P ＞ 0.05) between any two, different letter representation (the P ＜ of significant difference between any two
0.05)。
The biomembranous removing of E. coli mature is made by embodiment 15 leaf of Moringa ethanol extraction and Cortex Cinnamomi ethanol extraction
With
Escherichia coli form the biomembrane of maturation after cultivating 48h, by leaf of Moringa ethanol extraction and the Cortex Cinnamomi of variable concentrations
Ethanol extraction and their 1:1 (v/v) compound are added thereto, and set negative control simultaneously, continue to cultivate 24h；Then distinguish
Survey biofilm biomass, the generation amount of extracellular polysaccharide, calculate clearance rate.Result shows, these extracts are to Escherichia coli biofilm all
There is scavenging action (accompanying drawing 18)；Escherichia coli extracellular polysaccharide is also had the strongest Scavenging activity (accompanying drawing 19).Note: same letter
Represent that difference is not notable (P ＞ 0.05) between any two, different letter representation significant differences between any two (P ＜ 0.05).
The removing of vibrio parahaemolytious biofilm is made by embodiment 16 Cortex Cinnamomi ethanol extraction and Bulbus Allii ethanol extraction
With
Vibrio parahaemolytious forms the biomembrane of maturation after cultivating 48h, by leaf of Moringa ethanol extraction and the meat of variable concentrations
Osmanthus ethanol extraction and their 1:1 (v/v) compound are added thereto, and set negative control simultaneously, continue to cultivate 24h；Then divide
Do not survey the generation amount of biofilm biomass, extracellular polysaccharide, calculate clearance rate.Result shows, these extracts are biological to vibrio parahaemolytious
Film has scavenging action (accompanying drawing 20)；Vibrio parahaemolytious extracellular polysaccharide is also had the strongest Scavenging activity (accompanying drawing 21).Note: phase
Not notable (P ＞ 0.05) with letter representation difference between any two, different letter representation significant differences between any two (P ＜ 0.05).
Embodiment 17 CLSM observes extract to three kinds of biomembranous inhibitory action of bacterium
Laser confocal scanning microscope is except observing the plane picture of bacterial biof iotalm, additionally it is possible to biomembranous
Stereochemical structure carries out 3-D scanning, and can also observe extracellular polysaccharide intuitively under the effect of fluorescent dye and cell is many
Few.Observed formation and the suppression situation of bacterial biof iotalm by CLSM, its result is as shown in figs. 22-24.Note: a, b are matched group,
C, d are experimental group.
As shown in Figure 22, in a, bacterial accumulation is more apparent, and polyoses content is more and is mainly distributed on around antibacterial, and in c
Polysaccharide is evenly distributed, Multiple drug resistance relative distribution；B with d then clearly demonstrates that the polysaccharide thickness of matched group is than experimental group
Want thickness.Therefore, the Bulbus Allii extract of 2 × MIC concentration processes staphylococcus aureus except killing the extracellular of a part,
The secretion of extracellular polysaccharide can also be suppressed, thus reduce biomembranous formation.
As shown in Figure 23, substantially many than in c of the polysaccharide in a, and also polysaccharide is assembled obvious and surrounds antibacterial,
Polysaccharide and cell in c disperse the most very much；By the comparison of b and d, the change of polyoses content can be found out the most intuitively.Say
The Cortex Cinnamomi extract of bright 2 × MIC concentration has the strongest inhibitory action to colibacillary biofilm formation.
As shown in Figure 24, found by the comparison of a Yu c and b Yu d, at the Cortex Cinnamomi extract through 2 × MIC concentration
After reason, the quantity showed increased of dead cell, and polyoses content significantly reduces, and illustrates that vibrio parahaemolytious is had by Cortex Cinnamomi stronger
Killing action, and the generation of extracellular polysaccharide can be efficiently reduced.
Embodiment 18 CLSM observes extract to three kinds of biomembranous Scavenging activity of bacterium
After the biomembrane that extract-treated is ripe, the change of its structure is as shown in accompanying drawing 25-27.Note: a, b are matched group,
C, d are experimental group.
As shown in Figure 25, in a, staphylococcus aureus defines biomembrane after cultivating 48h, and substantial amounts of extracellular polysaccharide is assembled
Around antibacterial, although and c has substantial amounts of antibacterial, but its polyoses content is considerably less, and is all dead antibacterial, also can compared with b with d
Enough find that experimental group polyoses content decreases a lot than matched group, illustrates that Bulbus Allii extract has gone out and can kill antibacterial, additionally it is possible to
Remove biomembranous polysaccharide to a certain extent.
Figure 26 is to process the contrast before and after Escherichia coli biofilm, as seen from the figure, the biology of matched group for Cortex Cinnamomi extract
Film grow ratio is more uniform, polysaccharide is wrapped in antibacterial, and is evenly distributed.And after processing, Multiple drug resistance ratio is relatively decentralized, many
Sugar content reduces, dead bacterial number showed increased.Illustrate that Cortex Cinnamomi still has stronger Scavenging activity to ripe biomembrane.
Figure 27 is to process the contrast before and after vibrio parahaemolytious biomembrane, as seen from the figure, in matched group for Cortex Cinnamomi extract
Vibrio parahaemolytious biofilm formation amount is bigger, polysaccharide the biomembrane being wrapped in, overall in limegreen, illustrates mainly by many
Sugar composition, and dead antibacterial is less.And in experimental group, major part biomembrane has been cleared by, and color becomes orange-yellow, says
Bright extracellular polysaccharide decreases and the quantity of dead antibacterial increases.The biomembrane of vibrio parahaemolytious is had good by these explanation Cortex Cinnamomis
Scavenging activity.In Figure 22-27, a, b, c, d are mainly used in embodying contrast effect.
Claims (7)
1. spice extract application in the suppression of food-borne pathogens biomembrane and removing, described spice extract is fragrant
Pungent material alcohol extraction thing or spice water extract.
Application the most according to claim 1, it is characterised in that described spice extract be Bulbus Allii, Cortex Cinnamomi, leaf of Moringa,
Moringa stem, Flos Caryophylli extract in one or both.
Application the most according to claim 1, it is characterised in that described food-borne pathogens is staphylococcus aureus or big
Enterobacteria or vibrio parahaemolytious.
Application the most according to claim 1, it is characterised in that described spice extract is prepared as follows: fragrant pungent
The standby raw material break process of material obtains material slag, adds extracting solution by solid-liquid ratio 1:6～12, extracts with ultrasonic assistant, and temperature is 40
～60 DEG C, extraction time is 0.5～1.5h, decompression sucking filtration after concentrate with Rotary Evaporators, rotary evaporation condition be 50～
60 DEG C, proceed to after tool fills in test tube constant volume and get final product.
Application the most according to claim 4, it is characterised in that described extracting solution is ethanol or the water of 80%.
Application the most according to claim 1, it is characterised in that described spice extract is to food-borne pathogens biomembrane
Suppression be embodied as suppress the biomembranous formation of food-borne pathogens, suppression metabolic activity and suppression produce extracellular polysaccharide,
And spice extract is 6.25mg/mL-12.5mg/mL to the minimal inhibitory concentration of food-borne pathogens.
Application the most according to claim 1, it is characterised in that described spice extract is to food-borne pathogens biomembrane
Removing be embodied as entering in ripe biomembrane and kill wherein part food-borne pathogens and decomposing organism film
Extracellular polysaccharide.
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