CN106360250A - Applications of lectin-modified anchovy antibacterial peptide liposome in inhibition of Listeria monocytogenes and biofilm thereof - Google Patents
Applications of lectin-modified anchovy antibacterial peptide liposome in inhibition of Listeria monocytogenes and biofilm thereof Download PDFInfo
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- CN106360250A CN106360250A CN201510423083.XA CN201510423083A CN106360250A CN 106360250 A CN106360250 A CN 106360250A CN 201510423083 A CN201510423083 A CN 201510423083A CN 106360250 A CN106360250 A CN 106360250A
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- antibacterial peptide
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- listeria monocytogenes
- lectin
- anchovy
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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
- 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
- A23L3/3571—Microorganisms; Enzymes
Abstract
The invention discloses applications of a lectin-modified anchovy antibacterial peptide liposome in inhibition of Listeria monocytogenes and a biofilm thereof, and belongs to the technical field of food microorganism control. According to the present invention, the lectin-modified anchovy antibacterial peptide liposome is prepared according to the specific affinity between the lectin and the saccharide unit in the capsule extracellular polysaccharide matrix, the Listeria monocytogenes biofilm model is established, and the inhibition effect of the lectin-modified anchovy antibacterial peptide liposome on the Listeria monocytogenes and the biofilm thereof is evaluated through the sterilization curve, the colony counting and the crystal violet staining, wherein the results show that the lectin-modified anchovy antibacterial peptide liposome provides the sterilization effect on the Listeria monocytogenes, can interfere the formation of the biofilm, and can eliminate the formed biofilm, and the effect is concentration dependent, such that the lectin-modified anchovy antibacterial peptide liposome can be used for inhibiting or clearing the Listeria monocytogenes and the biofilm thereof in the food processing and storage environments.
Description
Technical field
The invention belongs to food microorganisms control technology field, it is related to application in suppression Listeria monocytogenes and its biofilm for the Phytoagglutinin modified type fish antibacterial peptide liposome.
Background technology
Listeria monocytogenes are one of four big food-borne pathogens of World Health Organization's definition.This bacterium can be entered in people and animals' body by the food of pollution, causes the symptoms such as septicemia, meningitiss, miscarriage and monocytosis, fatality rate reaches 20～30%.Repeatedly the clinical disease problem that the food pollution of this bacterium initiation leads in the states such as the U.S., France, Canada.Listeria monocytogenes can be attached on food or food processing equipment surface, forms biofilm.Biofilm is a kind of growth patterns in biology or inanimate surfaces for microbial cell corresponding with planktonic cells, is that polysaccharide, fibrin and lipid protein of cell secretion etc. have protectiveness and its own is wrapped up and the structural microbiologic population of formation by the poly substrate of adsorptivity.Established biofilm or the hiding-place of other various pathogens and putrefactive microorganisms, the mutual inductance effect between bacterium colony causes the collaborative symbiosis of each microorganism, the pollution of increase food itself and processing environment or recontamination risk.Envelope state and difference on cellularity and biological characteristicses for the state Listeria monocytogenes of swimming, lead to that the former is more higher than the latter resistance, harm is more serious and is more difficult to remove.
Chinese scholars have carried out related exploration in the control method of Listeria monocytogenes and its biofilm.However, these methods mainly adopt chemosterilant, natural essential oil, antibacterial peptide, phage and physical method.Can these methods be applied to field of food needs to consider its safety, effectiveness and economy.Pathogenic bacterium easily produce drug resistance to chemosterilant, and the genotoxic potential of chemicals and its residual can reduce foodsafety.The stronger natural flavour mountaineous sensory properties that may interfere with food itself of quintessence oil class, and its hydrophobic property can hinder its diffusion internally from biofilm surface.The safety that phage is applied in food still needs to evaluate, and heavy dose of use cost is higher and appearance that may result in phage resistant strains.Some physical means such as ultrasound wave, high-pressure pulse electric etc., relatively costly and have certain limitation.Therefore, it is necessary to exploitation new, safe efficient, have potential food service industry using value control/removing Listeria monocytogenes and its biofilm method.
Antibacterial peptide is the small molecule small peptide that a class has broad spectrum antibiotic activity.Antibacterial peptide has the bactericidal mechanism different from conventional antibiotic, is not likely to produce pathogenic bacteria resistance to drugs and cross tolerance.Small part antibacterial peptide has been shown to have the effect of suppression Listeria monocytogenes and its biofilm, such as peptide1037 (de la fuente-n ú ez c, korolik v, bains m,
et al. inhibition of bacterial biofilm formation and swarming motility by a
small synthetic cationic peptide[j]. antimicrob agents ch, 2012, 56(5):
2696-2704.).Liposome embedded type antibacterial peptide has the characteristics that targeting, controlled capability, cellular affinity and histocompatibility.Biofilm contains a large amount of extracellular polysaccharide substrate, combines effect based on the specific sugar of agglutinin, on medicinal liposome connect agglutinin, achievable study on targeting of liposome act on oral cavity bacterium biofilm target (jones m n, francis s e,
hutchinson f j, et al. targeting and delivery of bactericide to adsorbed oral
bacteria by use of proteoliposomes[j]. bba-biomembranes, 1993, 1147(2):
251-261.).However, in prior art, having no using food source especially source of fish antibacterial peptide as the thing that is embedded, build agglutinin surface modification type antibacterial peptide liposome, suppression or the relevant report removing Listeria monocytogenes biofilm.
Content of the invention
The purpose of the present invention is the food pollution for solving the problems, such as food processing field of storage Listeria monocytogenes and its biofilm causes, and provides a kind of purposes in suppression Listeria monocytogenes and its biofilm for Phytoagglutinin modified type fish antibacterial peptide liposome.
For achieving the above object, the present invention is with fish antibacterial peptide (glarclagtl) for the thing that is embedded, prepare Phytoagglutinin modified fish antibacterial peptide liposome, investigate its inhibition to the state Listeria monocytogenes that swim, set up Listeria monocytogenes external biological by membrane modle, the inhibitory action to Listeria monocytogenes biofilm for the Phytoagglutinin modified type fish antibacterial peptide liposome is evaluated by crystal violet staining assay.
It is a discovery of the invention that the Phytoagglutinin modified type fish antibacterial peptide liposome of tetra- kinds of variable concentrations of 0.5mic, 1mic, 2mic and 3mic all has bactericidal action to Listeria monocytogenes, and bactericidal effect improves with the increase of antibacterial peptide concentration.In the culture starting stage, add the Phytoagglutinin modified type fish antibacterial peptide liposome of above-mentioned four kinds of concentration, the formation to Listeria monocytogenes biofilm has the effect of significantly inhibiting.Biofilm forming amount reduces with the increase of antibacterial peptide liposome concentration.The Phytoagglutinin modified type fish antibacterial peptide liposome of variable concentrations has notable scavenging action to established Listeria monocytogenes biofilm.Its elimination effect with the increase of antibacterial peptide liposome concentration, the prolongation of checkout time and increase.
In sum, the effect that Phytoagglutinin modified type fish antibacterial peptide liposome has suppression and removes Listeria monocytogenes and its biofilm.
Above-described agglutinin is one kind of phytohemagglutinin；It is preferably wheat germ agglutinin and with ConA.
The Amino acid group of above-described fish antibacterial peptide becomes glarclagtl
(gly-leu-ala-arg-cys-leu-ala-gly-thr-leu), its molecular weight is 973.11 da.
Compared with prior art, the advantage of the inventive method is embodied in:
1. in the present invention, antibacterial peptide used and agglutinin are food sources, safe and effective can be applied to field of food；
2. in the present invention, Phytoagglutinin modified type fish antibacterial peptide liposome has, to Listeria monocytogenes and its biofilm, the effect of significantly inhibiting；
3. the present invention is with Phytoagglutinin modified type fish antibacterial peptide liposome as core technology, specificity affinity interaction using the glucide in agglutinin and biofilm Extracellular polymers substrate, it is not likely to produce drug resistance, the controlled capability of liposome, cell warm property feature in conjunction with antibacterial peptide, the targeting of achievable Listeria monocytogenes biofilm controls.
Brief description
The killing curve to Listeria monocytogenes for Fig. 1 Phytoagglutinin modified type fish antibacterial peptide liposome.
The inhibitory action that Fig. 2 Phytoagglutinin modified type fish antibacterial peptide liposome is formed to Listeria monocytogenes biofilm.
The scavenging action to the Listeria monocytogenes biofilm that 24 h cultures are formed for Fig. 3 Phytoagglutinin modified type fish antibacterial peptide liposome.
Specific embodiment
The invention anti-Listeria monocytogenes and its biofilm activity research are carried out using Phytoagglutinin modified type fish antibacterial peptide liposome, apply in field of food for it and lay the foundation.The following is the effect example that Phytoagglutinin modified type fish antibacterial peptide liposome suppresses Listeria monocytogenes biofilm to be formed.
Embodiment 1: the preparation of Phytoagglutinin modified type fish antibacterial peptide liposome
PHOSPHATIDYL ETHANOLAMINE (600 mg) is dissolved in 60 ml chloroforms, adds 940 mg glutaric anhydrides, 150 μ l pyridines to react 5 h at 20 DEG C.Reactant liquor obtains glutaryl PHOSPHATIDYL ETHANOLAMINE using silica gel post separation.Take 10 mg lyophilizing glutaryl PHOSPHATIDYL ETHANOLAMINE to be scattered in 150 μ l, add 2 ml 0.15 mol/l sodium chloride, supersound process 15 s.The ph of reactant liquor is adjusted to 3.5, adds 20 mg1- ethyl -3- (3- dimethylamino-propyl) carbodiimides, concussion is uniformly.After reacting 5 min, add the pbs solution of the agglutinin of 1 ml 20 mg/ml, 4 DEG C of reaction 2 h.Product flow deionized water is dialysed 24 h, then with pbs buffer dialysis 48h, freezes in obtaining Phytoagglutinin modified PHOSPHATIDYL ETHANOLAMINE.Appropriate PHOSPHATIDYL ETHANOLAMINE fat and cholesterol is taken to be dissolved in chloroform, rotary evaporation removes organic solvent.Add 2.048 mg/ml antibacterial peptide solutions (being dissolved in pbs buffer), ice-bath ultrasonic process 5 min, obtain final product Phytoagglutinin modified insulin liposome.
Embodiment 2: the mensure to Listeria monocytogenes minimal inhibitory concentration and minimal bactericidal concentration for the Phytoagglutinin modified type fish antibacterial peptide liposome
Phytoagglutinin modified insulin liposome solution is degerming through 0.22 μm of membrane filtration, and using sterilizing pbs buffer doubling dilution.Listeria monocytogenes bacterium solution is inoculated in 37 DEG C of overnight incubation in aseptic tsb culture fluid, gained thalline resuspended to od600Between 0.1 ~ 0.3.Successively the antibacterial peptide liposome solution of 100 μ ltsb culture medium, the tested bacterium solution of 100 μ l and 50 μ l variable concentrations is added to flat 96 orifice plates of sterilizing.37 DEG C of culture 18 h.Microplate reader measures every in the hole od600Value.Matched group replaces antibacterial peptide liposome suspension using the modification type lipid liposome that equivalent does not encapsulate antibacterial peptide.Compared with initial value, od600The Cmin of the peptide not being further added by is the minimal inhibitory concentration (mic) of antibacterial peptide liposome.Mixed liquor is taken to be inoculated in solid medium from 96 orifice plates successively, 37 DEG C of culture 24 h, observation has or not bacterium colony and is formed, and the antibacterial peptide liposome concentration in the corresponding test tube that no bacterium colony is formed is i.e. minimal inhibitory concentration (mbc).
Embodiment 3: the sterilization kinetics to Listeria monocytogenes for the Phytoagglutinin modified type fish antibacterial peptide liposome
Listeria monocytogenes bacterium solution is inoculated in 37 DEG C of overnight incubation in aseptic tsb culture fluid, using resuspended after pbs buffer solution 3 times be to 1.0 × 107cfu/ml.Phytoagglutinin modified type fish antibacterial peptide liposome (final concentration of 0.5mic, 1mic, 2mic and 3mic) is added in bacteria suspension.Bacterial growth control group adds isopyknic pbs buffer.Mix and cultivate after 37 DEG C, take the bacteria suspension of 100 μ l every 0.5 h, after carrying out suitable 10 times of gradient dilutions, take 100 μ l to coat on solid medium.Culture dish is inverted in biochemical cultivation case, counts after 37 DEG C of culture 24 h.3 repetitions of Setup Experiments, results averaged.
Embodiment 4: the suppression that Phytoagglutinin modified type fish antibacterial peptide liposome is formed to Listeria monocytogenes biofilm
Sequentially add the antibacterial peptide liposome suspension of 100 μ ltsb culture medium, 100 μ l bacteria suspensions and variable concentrations, 37 DEG C of cultures to the sterilizing every hole of flat 96 orifice plates, respectively after culture 1,2,4,6,8,10,12,24 h, discard culture fluid in plate.Sterilizing pbs buffer solution for cleaning 3 times, 30 DEG C of drying 30 min.45 min are dyeed using 0.2% crystal violet 200 μ l, 30 DEG C of drying 30 min add the ethanol of 200 μ l 95% after being dried, microplate reader is surveyed its absorbance at 595 nm, tested parallel 3 times.
Embodiment 5: the removing to Listeria monocytogenes biofilm for the Phytoagglutinin modified type fish antibacterial peptide liposome
Sequentially add 100 μ l tsb culture medium, 100 μ l bacteria suspensions, 37 DEG C of culture 24 h to the sterilizing every hole of flat 96 orifice plates.Discard culture fluid, sterilizing pbs buffer solution for cleaning 3 times, every hole adds the antibacterial peptide liposome suspension of 200 μ l variable concentrations.Effect 1,2,4,6,8,10,12, after 24h, discard antibacterial peptide liposome.Sterilizing pbs buffer solution for cleaning 3 times, fixes 30 min for 55 DEG C.45 min are dyeed using 0.2% crystal violet 200 μ l, after being dried, adds the ethanol of 200 μ l 95%, microplate reader surveys its absorbance at 595 nm, test parallel 3 times.
Claims (5)
1. application in suppression Listeria monocytogenes for the Phytoagglutinin modified type fish antibacterial peptide liposome.
2. Phytoagglutinin modified type fish antibacterial peptide liposome is intervening the application during Listeria monocytogenes biofilm is formed.
3. application in removing established Listeria monocytogenes biofilm for the Phytoagglutinin modified type fish antibacterial peptide liposome.
4. according to the purposes described in claims 1 to 3 any one it is characterised in that: described agglutinin be food sources phytohemagglutinin one kind；It is preferably wheat germ agglutinin and with ConA.
5. according to the purposes described in claims 1 to 3 any one it is characterised in that: described fish antibacterial peptide amino acid sequence be glarclagtl.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111406872A (en) * | 2020-03-10 | 2020-07-14 | 集美大学 | Application of tegillarca granosa hemoglobin antibacterial peptide in food preservation and freshness keeping |
| CN112120140A (en) * | 2019-06-25 | 2020-12-25 | 中国科学院大连化学物理研究所 | Engraulis japonicus Temminck et Schlegel antibacterial substance, its preparation method and its application in food preservation |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103936832A (en) * | 2014-04-23 | 2014-07-23 | 江南大学 | Antibacterial peptide derived from anchovy cooking waste liquid and antibacterial peptide separation method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103936832A (en) * | 2014-04-23 | 2014-07-23 | 江南大学 | Antibacterial peptide derived from anchovy cooking waste liquid and antibacterial peptide separation method |
Non-Patent Citations (5)
| Title |
|---|
| WENTING TANG等: "New Cationic Antimicrobial Peptide Screened from Boiled-Dried Anchovies by Immobilized Bacterial Membrane Liposome Chromatography", 《JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY》 * |
| WENTING TANG等: "Targeted separation of antibacterial peptide from protein hydrolysate", 《FOOD CHEMISTRY》 * |
| 唐文婷: "基于肽-膜互相作用的模拟细胞膜法筛选抗菌肽的研究", 《中国博士学位论文全文数据库 工程科技 I辑》 * |
| 唐文婷: "基于肽-膜相互作用的模拟细胞膜法筛选抗菌肽的研究", 《中国博识学位论文全文库工程科技I辑》 * |
| 艾丽思等: "抗菌肽及其在口腔领域应用前景", 《中国实用口腔杂质》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112120140A (en) * | 2019-06-25 | 2020-12-25 | 中国科学院大连化学物理研究所 | Engraulis japonicus Temminck et Schlegel antibacterial substance, its preparation method and its application in food preservation |
| CN111406872A (en) * | 2020-03-10 | 2020-07-14 | 集美大学 | Application of tegillarca granosa hemoglobin antibacterial peptide in food preservation and freshness keeping |
| CN111406872B (en) * | 2020-03-10 | 2022-07-19 | 集美大学 | Application of tegillarca granosa hemoglobin antibacterial peptide in food preservation and freshness keeping |
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