CN102318873A - Bacterial population sensing inhibitor and screening method thereof - Google Patents
Bacterial population sensing inhibitor and screening method thereof Download PDFInfo
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- CN102318873A CN102318873A CN201110163808A CN201110163808A CN102318873A CN 102318873 A CN102318873 A CN 102318873A CN 201110163808 A CN201110163808 A CN 201110163808A CN 201110163808 A CN201110163808 A CN 201110163808A CN 102318873 A CN102318873 A CN 102318873A
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Abstract
The invention relates to a bacterial population sensing inhibitor and a screening method thereof. The screening method is characterized by comprising the following steps of: drying algae and smashing; adding an ethanol solution for extracting; and detecting an extracting solution with bacterium biosensor chromobacterium CV026 and caproyl homoserine lactone, wherein the extracting solution is the bacterial population sensing inhibitor if a detection result is positive. The inhibitor screening method is quick and effective, and the bacterial population sensing inhibitor can be efficiently screened from marine algae. The bacterial population sensing inhibitor screened with the method can be applied to preservation of fresh foods or products, and is natural and nontoxic and has a better preservation effect in comparison to the conventional chemical synthesis inhibitor. The screening method is suitable for industrial production. The yield of the prepared inhibitor can well meet practical application.
Description
Technical field
The invention belongs to the food preservative technology field, be specifically related to a kind of bacterial community induction inhibitor and screening technique thereof.
Background technology
" quorum sensing " of bacterium (Quorum Sensing, be called for short QS) promptly depends on the induction (Sensing) that could take place when number of bacteria (Quorum) reaches certain density.When the bacterial number in the specific environment increases, can activate the bacterium target gene expression, represent the behavioural characteristic that makes new advances, as bioluminescence, plasmid shift, the sprouting of the generation of virulence factor, spore and biomembranous formation etc.The decay process of food receives spoilage organisms QS system regulation, and the process that with the colony induction signaling molecule is novel targets regulation and control food spoilage is that food storage is fresh-keeping provides a kind of New Policy.
Combine with receptor protein to influence the main direction that the QS system is quorum sensing inhibitor (QSI) research through the interfering signal molecule, and the source of inhibitor mainly comprises artificial synthetic and naturally occurring micromolecular compound.Consider that from the angle of food security in recent years, natural population's induction inhibitor receives much concern.
Patent about quorum sensing mainly concentrates on the detection method of quorum sensing and the gene regulation aspect of quorum sensing; Patent (a kind of method that detects gram negative bacteria quorum sensing inhibitor wherein; 200910025868.6) reported and the detection method of quorum sensing inhibitor mainly detected microbe-derived quorum sensing inhibitor.But microbe-derived quorum sensing inhibitor has limitation in Application in Food, and microbial metabolic products is complicated, often produces harmful metabolite, and food is produced pollution or causes food poisoning.Therefore, be badly in need of obtaining a kind of bacterial community induction inhibitor that can in field of food, use, to fill the blank of China about marine alga bacterial community induction inhibitor.
Summary of the invention
The purpose of this invention is to provide a kind of bacterial community induction inhibitor and screening technique thereof, to overcome the deficiency of prior art.
The present invention separates preparation bacterial community induction inhibitor from the marine algae of inexhaustible desirable feedstock, its product has natural, safe characteristics, and China's marine algae resource is abundant, is more conducive to the suitability for industrialized production of product of the present invention.
Bacterial community induction inhibitor of the present invention is the marine alga ethanol extract to bacterium living beings inductor chromabacterium biolaceum CV026 and caproyl homoserine lactone test positive.
The screening technique of bacterial community induction inhibitor of the present invention is following: at first with after the marine alga drying and crushing; Adding ethanolic solution extracts; Extract detects with bacterium living beings inductor chromabacterium biolaceum CV026 and caproyl homoserine lactone, and the extract of test positive is a bacterial community induction inhibitor of the present invention.
Above-mentioned detection method can use following method to substitute: the mini-Tn5 mutant chromabacterium biolaceum CV026 of wild strain Chromobacterium violaceum ATCC 31532 is spent the night after twice of the activation; Be inoculated in the fresh LB meat soup that contains 20 μ g/mL kanamycins in 2% ratio, behind the 150r/min shaken cultivation 16-18h, the LB culture medium that contains 10 μ mol/L acidylate homoserine lactone C6-HSL, 0.8g/100mL agar with 100mL mixes; Fall dull and stereotyped; Wait to solidify the back and use the card punch punching of diameter, above-mentioned seaweed extracted liquor is added cultivate in the datum hole, flat board is placed 27-30 ℃ constant incubator incubated overnight 24h as 6mm; Observe phenomena can judge whether bacterial community is responded to inhibitor to seaweed extracted liquor; If having added around the hole of seaweed extracted liquor is purple, promptly testing result is negative, shoals or takes off if added around the hole of seaweed extracted liquor purple; Then testing result is positive, and this seaweed extracted liquor is a bacterial community induction inhibitor.
Described marine algae is green alga, brown alga, red algae etc.
The positive bacteria quorum sensing inhibitor that detects is for being the extract of blunt top spirulina, decomposite leaf sargassum or undaria pinnitafida.
Inhibitor screening method of the present invention is effective fast, can from marine algae, filter out bacterial community induction inhibitor efficiently.The bacterial community induction inhibitor that the present invention filters out can be applicable to the anti-corrosive fresh-keeping of FF or goods, and than existing chemical synthesis inhibitor Nantural non-toxic evil, fresh-keeping effect is better.Screening technique of the present invention is fit to suitability for industrialized production, and the inhibitor output of preparation can be good at satisfying practical application.
The specific embodiment
Screening technique of the present invention at first sieves grinding after the marine alga freeze drying; Get seaweed meal; The solid-liquid ratio of pressing 1: 10 (g/mL) is with 75%-85% ethanol ultrasonic Extraction 30min, and the centrifugal residue that removes is filtrated and got the marine alga crude extract through Rotary Evaporators evaporate to dryness ethanol; Crude extract successively promptly gets seaweed extracted liquor with benzinum, ethyl acetate extraction water intaking mutually; This extract detects with bacterium living beings inductor chromabacterium biolaceum CV026 and caproyl homoserine lactone through 0.22 μ m membrane filtration degerming again, and the extract of test positive is a bacterial community induction inhibitor of the present invention.
Above-mentioned bacterium living beings inductor chromabacterium biolaceum CV026 and caproyl homoserine lactone detect with reference to Chinese invention patent: a kind of method that detects gram negative bacteria quorum sensing inhibitor, application number 200910025868.6 carries out.
Above-mentioned detection method can also use following method to substitute: bacterial strain chromabacterium biolaceum CV026 is spent the night after twice of the activation; Be inoculated in the fresh LB meat soup that contains 20 μ g/mL kanamycins in 2% ratio, behind the 150r/min shaken cultivation 16-18h, the LB culture medium that contains 10 μ mol/L acidylate homoserine lactone C6-HSL, 0.8g/100mL agar with 100mL mixes; Fall dull and stereotyped; Wait to solidify the back and use the card punch punching of diameter, above-mentioned seaweed extracted liquor is added cultivate in the datum hole, flat board is placed 27-30 ℃ constant incubator incubated overnight 24h as 6mm; Observe phenomena can judge whether bacterial community is responded to inhibitor to seaweed extracted liquor; If having added around the cultivation datum hole of seaweed extracted liquor is purple, promptly testing result is negative, shoals or takes off if added around the cultivation datum hole of seaweed extracted liquor purple; Then testing result is positive, shows that this seaweed extracted liquor is a bacterial community induction inhibitor.
Wherein bacterial strain chromabacterium biolaceum CV026 is the mini-Tn5 mutant of wild strain Chromobacte rium violaceum ATCC31532, also is documented in application number and is in 200910025868.6 the patent of invention.
Below in conjunction with embodiment screening technique of the present invention is described in detail:
Embodiment 1
Blunt top spirulina is pulverized through freeze drying, crosses 40 mesh sieves, gets the 100g powder; Add 75% ethanol 1000mL, ultrasonic Extraction 30min removes by filter residue, and residue repeats to extract twice; Merge three filtratings through Rotary Evaporators evaporate to dryness (vacuum 0.1kPa, 30 ℃ of temperature), extract concentrates (13.25g), adds with volume benzinum and distilled water extraction; Discard the benzinum phase, water is used ethyl acetate extraction again, discards ethyl acetate; Water is used the Rotary Evaporators evaporate to dryness, uses distilled water to be made into the seaweed extracted liquor of mass and size concentration as 0.125%-1%, and this extract is through 0.22 μ m membrane filtration degerming; Detect through bacterium living beings inductor chromabacterium biolaceum CV026 and caproyl homoserine lactone, this extract testing result is positive, confirms as bacterial community induction inhibitor-blunt top spirulina inhibitor.
In order to verify the validity of the inventive method, it is that the inhibitor aqueous solution of the present embodiment of 0.125%-1% soaks the back and takes out preservation as experimental group that fresh shrimp is put into concentration, totally 3 parallel; Simultaneously will be with a collection of bright shrimp with the distilled water immersion that will not add inhibitor as control group, also be 3 parallel; Control group and experimental group all are placed on the preservation down of same ice temperature, survey the shelf-life.The result shows that the shrimp shelf-life of control group is merely 4 days, and the shrimp shelf-life of immersion group extract obtains prolonging, and after 6 days, also has good freshness.Simultaneously, do not have the harmful chemical that inhibitor produced of chemical synthesis in the shrimp of liquid chromatographic detection demonstration experimental group, can satisfy the requirement of food security.
Embodiment 2
The decomposite leaf sargassum is pulverized through freeze drying, crosses 40 mesh sieves, gets the 100g powder; Add 80% ethanol 1000mL, ultrasonic Extraction 30min removes by filter residue, and residue repeats to extract twice; Merge three filtratings through the Rotary Evaporators evaporate to dryness, extract concentrates (3.82g), successively uses benzinum, ethyl acetate extraction; The water intaking phase, water is used the Rotary Evaporators evaporate to dryness, uses distilled water to be made into the seaweed extracted liquor of mass and size concentration as 0.1g/mL-1g/mL; Detect through bacterium living beings inductor chromabacterium biolaceum CV026 and caproyl homoserine lactone after the degerming, this extract testing result is positive, confirms as bacterial community induction inhibitor-decomposite leaf sargassum inhibitor.
In order to verify the validity of the inventive method, it is that the inhibitor aqueous solution of the present embodiment of 0.1g/mL-1g/mL soaks the back and takes out preservation as experimental group that fresh shrimp is put into concentration, totally 3 parallel; Simultaneously will be with a collection of bright shrimp with the distilled water immersion that will not add inhibitor as control group, also be 3 parallel; Control group and experimental group all are placed on the preservation down of same ice temperature, survey the shelf-life.The result shows that the shrimp shelf-life of control group is merely 4 days, and the shrimp shelf-life of immersion group extract obtains prolonging, and after 5 days, also has good freshness.Simultaneously, do not have the harmful chemical that inhibitor produced of chemical synthesis in the shrimp of liquid chromatographic detection demonstration experimental group, can satisfy the requirement of food security.
Embodiment 3
Undaria pinnitafida is pulverized through freeze drying, crosses 40 mesh sieves; Get the 100g powder, add 85% ethanol 1000mL, ultrasonic Extraction 30min; Make marine algae extract 15.32g, successively use benzinum, ethyl acetate extraction, the water intaking phase; Water is used the Rotary Evaporators evaporate to dryness, uses distilled water to be made into the seaweed extracted liquor of mass and size concentration as 0.01g/mL-1g/mL.Detect through bacterium living beings inductor chromabacterium biolaceum CV026 and caproyl homoserine lactone after the degerming, this extract testing result is positive, confirms as bacterial community induction inhibitor-undaria pinnitafida inhibitor.
In order to verify the validity of the inventive method, it is that the inhibitor aqueous solution of the present embodiment of 0.01g/mL-1g/mL soaks the back and takes out preservation as experimental group that fresh shrimp is put into concentration, totally 3 parallel; Simultaneously will be with a collection of bright shrimp with the distilled water immersion that will not add inhibitor as control group, also be 3 parallel; Control group and experimental group all are placed on the preservation down of same ice temperature, survey the shelf-life.The result shows that the shrimp shelf-life of control group is merely 4 days, and the shrimp shelf-life of immersion group extract obtains prolonging, and after 7 days, also has good freshness.Experimental result shows that the undaria pinnitafida extract can the more effective fresh-keeping effect that plays as inhibitor.
Claims (8)
1. a bacterial community induction inhibitor is the marine alga ethanol extract to bacterium living beings inductor chromabacterium biolaceum CV026 and caproyl homoserine lactone test positive.
2. the screening technique of the described bacterial community induction of claim 1 inhibitor is following: at first with after the marine alga drying and crushing; Adding ethanolic solution extracts; Then extract is detected with bacterium living beings inductor chromabacterium biolaceum CV026 and caproyl homoserine lactone, the positive extract of testing result is bacterial community induction inhibitor.
3. screening technique as claimed in claim 2; The detection method that it is characterized in that said extracted liquid is with following method replacement: the mini-Tn5 mutant chromabacterium biolaceum CV026 of wild strain Chromobacterium violaceum ATCC 31532 is spent the night after twice of the activation; Be inoculated in the fresh LB meat soup that contains 20 μ g/mL kanamycins in 2% ratio, behind the 150r/min shaken cultivation 16-18h, the LB culture medium that contains 10 μ mol/L acidylate homoserine lactone C6-HSL, 0.8g/100mL agar with 100mL mixes; Fall dull and stereotyped; Wait to solidify the back and use the card punch punching of diameter, above-mentioned seaweed extracted liquor is added cultivate in the datum hole, flat board is placed 27-30 ℃ constant incubator incubated overnight 24h as 6mm; Observe phenomena can judge whether bacterial community is responded to inhibitor to seaweed extracted liquor; If having added around the hole of seaweed extracted liquor is purple, promptly testing result is negative, shoals or takes off if added around the hole of seaweed extracted liquor purple; Then testing result is positive, and this seaweed extracted liquor is a bacterial community induction inhibitor.
4. screening technique as claimed in claim 2, the solid-liquid ratio g/mL that it is characterized in that above-mentioned marine alga and ethanolic solution is 1: 10.
5. like claim 2 or 4 described screening techniques, the concentration that it is characterized in that above-mentioned ethanolic solution is 75%-85%.
6. like claim 2 or 4 described screening techniques, it is characterized in that above-mentioned algae is brown alga, red algae or green alga.
7. the described bacterial community induction of claim 1 inhibitor is characterized in that described bacterial community induction inhibitor is blunt top spirulina, decomposite leaf sargassum or undaria pinnitafida ethanol extract.
8. the described inhibitor of claim 1 is applied to the anti-corrosive fresh-keeping of food.
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| CN201110163808A CN102318873A (en) | 2011-06-17 | 2011-06-17 | Bacterial population sensing inhibitor and screening method thereof |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104356099A (en) * | 2014-11-19 | 2015-02-18 | 郑州大学 | Homoserine lactone compounds as well as preparation methods and application thereof |
| CN105504001A (en) * | 2016-01-21 | 2016-04-20 | 福建农林大学 | Method for rapidly screening bacterial quorum sensing inhibitor (QSI) by utilizing luminescence method |
| CN105733828A (en) * | 2016-03-07 | 2016-07-06 | 浙江工商大学 | Medicine for alleviating municipal sewage pipeline blocking and application of medicine |
| CN107183146A (en) * | 2017-06-15 | 2017-09-22 | 中国农业大学 | Quorum quenching enzymes and bacteriocin joint anti-rot and fresh-keeping method |
| CN109479907A (en) * | 2018-11-30 | 2019-03-19 | 福建农林大学 | A kind of preparation method of natural fine bacteria quorum sensing inhibitor |
| CN109706111A (en) * | 2019-02-21 | 2019-05-03 | 中山大学 | The quick screening model and its construction method of P. aeruginosa bacteria quorum sensing system inhibitor |
| CN111838295A (en) * | 2020-08-07 | 2020-10-30 | 中国科学院天津工业生物技术研究所 | Rainbow trout fresh-keeping method based on quorum sensing inhibitor cassia twig extract |
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| CN101503730A (en) * | 2009-03-12 | 2009-08-12 | 中国药科大学 | Method for detecting gram negative bacteria quorum sensing inhibitor |
| CN101967134A (en) * | 2010-09-09 | 2011-02-09 | 中国海洋大学 | Compound separated and extracted from marine penicillium sp.QY013 and application thereof |
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2011
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| CN101503730A (en) * | 2009-03-12 | 2009-08-12 | 中国药科大学 | Method for detecting gram negative bacteria quorum sensing inhibitor |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104356099A (en) * | 2014-11-19 | 2015-02-18 | 郑州大学 | Homoserine lactone compounds as well as preparation methods and application thereof |
| CN105504001A (en) * | 2016-01-21 | 2016-04-20 | 福建农林大学 | Method for rapidly screening bacterial quorum sensing inhibitor (QSI) by utilizing luminescence method |
| CN105504001B (en) * | 2016-01-21 | 2019-07-09 | 福建农林大学 | A method of quickly screening quorum-quenching agent using luminescence method |
| CN105733828A (en) * | 2016-03-07 | 2016-07-06 | 浙江工商大学 | Medicine for alleviating municipal sewage pipeline blocking and application of medicine |
| CN105733828B (en) * | 2016-03-07 | 2018-04-27 | 浙江工商大学 | A kind of medicament for slowing down the blocking of municipal wastewater pipeline and its application |
| CN107183146A (en) * | 2017-06-15 | 2017-09-22 | 中国农业大学 | Quorum quenching enzymes and bacteriocin joint anti-rot and fresh-keeping method |
| CN109479907A (en) * | 2018-11-30 | 2019-03-19 | 福建农林大学 | A kind of preparation method of natural fine bacteria quorum sensing inhibitor |
| CN109706111A (en) * | 2019-02-21 | 2019-05-03 | 中山大学 | The quick screening model and its construction method of P. aeruginosa bacteria quorum sensing system inhibitor |
| CN109706111B (en) * | 2019-02-21 | 2023-09-29 | 中山大学 | Rapid screening model of pseudomonas aeruginosa quorum sensing system inhibitor and construction method thereof |
| CN111838295A (en) * | 2020-08-07 | 2020-10-30 | 中国科学院天津工业生物技术研究所 | Rainbow trout fresh-keeping method based on quorum sensing inhibitor cassia twig extract |
| CN111838295B (en) * | 2020-08-07 | 2023-07-04 | 中国科学院天津工业生物技术研究所 | Rainbow trout fresh-keeping method based on quorum sensing inhibitor cassia twig extract |
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Application publication date: 20120118 |