CN108753632A - A method of dissolving candida albicans biofilm extracellular matrix - Google Patents
A method of dissolving candida albicans biofilm extracellular matrix Download PDFInfo
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- CN108753632A CN108753632A CN201810545746.9A CN201810545746A CN108753632A CN 108753632 A CN108753632 A CN 108753632A CN 201810545746 A CN201810545746 A CN 201810545746A CN 108753632 A CN108753632 A CN 108753632A
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Abstract
A method of dissolving candida albicans biofilm extracellular matrix, enzyme solution is added into bacterium solution, then it reacts 1.5 ~ 2.5 hours for 25 DEG C, centrifugation is to discard enzyme solution after the completion of reaction, then decanoy acetaldehyde sodium solution is added, 37 DEG C reaction 20 ~ 28 hours after be added XTT solution, then under the conditions of being protected from light, in 37 DEG C react 1.5 ~ 2.5 hours；The bacterium solution is to generate the bacterium solution for the candida albicans for having biofilm.Sodium Houttuyfonate is the addition extract of Chinese herbal medicine cordate houttuynia, and the present invention utilizes the synergistic effect of Sodium Houttuyfonate and enzyme, directionally hydrolyzing candida albicans biofilm extracellular matrix components, to effectively improve antibacterial effect.
Description
Technical field
The invention belongs to biotechnologies, are related to a kind of method of dissolving candida albicans biofilm extracellular matrix, special
It is not related to a kind of method of Sodium Houttuyfonate and enzyme synergistic effect dissolving candida albicans biofilm extracellular matrix.
Background technology
Candida albicans is a kind of metatroph, usually survives in skin, mucous membrane, alimentary canal and the other organs of human body
In, when Abwehrkraft des Koepers reduces, Candida albicans will be bred, and when reaching a certain amount of, human body will fall ill, thus, Bai Nian
Pearl bacterium is a kind of human condition pathogenic fungus.
Candida albicans can improve the drug resistance of itself by forming biofilm.The born of the same parents of candida albicans biofilm secretion
Epimatrix ingredient is extremely complex, contains protein on the whole（Account for extracellular matrix dry weight 55%）, polysaccharide（Account for extracellular matrix dry weight
25%）, lipid（Account for extracellular matrix dry weight 15%）And nucleic acid（Account for extracellular matrix dry weight 5%）Equal macromolecular substances.
Currently, although conventional antifungal drug kind is relatively limited, target spot is clearer, is conducive to make combining for drug
It is utilized with the redevelopment with new drug.Traditional Chinese medicine monomer and its extract are abundant antimycotic treasure-houses, but are made in the prevalence of drug
With the disadvantage that target spot is not clear enough.
Invention content
Present invention aims at provide a kind of method of dissolving candida albicans biofilm extracellular matrix.
To achieve the above object, technical solution provided by the invention is：A kind of extracellular base of dissolving candida albicans biofilm
Enzyme solution is added into bacterium solution for the method for matter, then reacts 1.5 ~ 2.5 hours for 25 DEG C, is centrifuged after the completion of reaction to discard enzyme solution,
Then be added decanoy acetaldehyde sodium solution, 37 DEG C reaction 20 ~ 28 hours after be added XTT solution, then under the conditions of being protected from light, in 37 DEG C
Reaction 1.5 ~ 2.5 hours；The bacterium solution is to generate the bacterium solution for the candida albicans for having biofilm.
Preferably technical solution is：A concentration of 50 μ g/mL of the enzyme solution.
Preferably technical solution is：The enzyme of the enzyme solution be selected from lywallzyme, ribonuclease A, deoxyribonuclease I,
Protease XIV.
Preferably technical solution is：A concentration of 512 ~ 1024 μ g/mL of the decanoy acetaldehyde sodium solution.
Preferably technical solution is：The XTT solution is by 3,3'- [1- (phenylamino acyl group) -3,4- tetrazoles]-two (4-
Methoxyl group -6- nitros) benzene sulfonic acid sodium salt is dissolved in ringer's solution and obtains.
Description of the drawings
Attached drawing 1 is suctions of the SH of various concentration at OD492nms of the 50 μ g/mL of lywallzyme concentration to candida albicans SC5314
Shading value.
Attached drawing 2 is suctions of the SH of various concentration at OD492nms of the 50 μ g/mL of RNA enzyme concentration to candida albicans SC5314
Shading value.
Attached drawing 3 is suctions of the SH of various concentration at OD492nms of the 50 μ g/mL of DNA enzymatic concentration to candida albicans SC5314
Shading value.
Attached drawing 4 is the SH of various concentration at OD492nms of the 50 μ g/mL of proteinase K concentration to candida albicans SC5314
Absorbance value.
Attached drawing 5 is the SH of various concentration at OD492nms of the 50 μ g/mL of protease XIV concentration to candida albicans SC5314
Absorbance value.
Attached drawing 6 is the SH of various concentration at OD492nms of the 50 μ g/mL of aminohexose enzyme concentration to candida albicans SC5314
Absorbance value.
Attached drawing 7 is the SH of various concentration at OD492nms of the 50 μ g/mL of uronic acid enzyme concentration to candida albicans SC5314
Absorbance value.
Since above-mentioned technical proposal is used, the present invention this have the advantage that compared with prior art：
Sodium Houttuyfonate is the addition extract of Chinese herbal medicine cordate houttuynia, and the present invention utilizes the synergistic effect of Sodium Houttuyfonate and enzyme,
Directionally hydrolyzing candida albicans biofilm extracellular matrix components, to effectively improve antibacterial effect.
Specific implementation mode
Illustrate that embodiments of the present invention, those skilled in the art can be by this explanations by particular specific embodiment below
Content disclosed by book understands other advantages and effect of the present invention easily.
Embodiment 1 ~ 8：A method of dissolving candida albicans biofilm extracellular matrix
1. experiment material and instrument
1.1 bacterial strain：ATCC reference cultures：Candida albicans（Candida albicans,C.albicans）SC5314.
1.2 antifungal drug：Sodium Houttuyfonate（SH）.
1.3 main agents：Deoxyribonuclease I（DNase I）（Article No. D5025）, ribonuclease A（RNase A）
（R6513）, β-n-acetylglucosamine glycosides enzyme（Article No. A2264）, Proteinase K（Article No. P6516）, protease XIV（Article No.
P5147）, lywallzyme（Article No. L4025）, GRD beta-glucuronidase（Article No. G7017）（Whole enzymes are all from sigma）, DEPC water,
SDS。
1.4 main solutions are prepared：RPMI-1640 culture mediums, SDA culture mediums, sabouraud culture medium, the XTT solution are will
- two (4- methoxyl group -6- nitros) benzene sulfonic acid sodium salts of 3,3'- [1- (phenylamino acyl group) -3,4- tetrazoles] are dissolved in ringer's solution and obtain.
1% SDS：0.1 g lauryl sodium sulfate dissolves in 10 ml DEPC water is stored in room temperature.0.5% SDS：0.05 g 12
Sodium alkyl sulfate is dissolved in room temperature in 10 ml DEPC water.
1.5 key instrument：Microtest plate（96 holes）（Corning companies of the U.S.）；Micro sample adding appliance（Finland
Finnpette companies）；Superpurgative working table, constant incubator（Shanghai Bo Xun industrial corporations）；Ai Kepu ultrapure water systems（Weight
Celebrating chin or cheek ocean）；Precise electronic balance（Shanghai is more flat）；SpectraMax M2e multi-function microplate readers（The U.S. limited public affairs of paddy molecule instrument
Department）.
2. experimental method
Bacterial concentration is adjusted to 2 × 10 by white read after type strain SC5314 is activated6CFU/mL takes the RPMI-1640 of 100 μ L to train
Foster base adds same volume bacterium solution in 37 DEG C of 96 orifice plate constant-temperature incubation, forms biofilm for 24 hours, is then slowly inhaled and is abandoned with micro sample adding appliance
100 μ L supernatants（Except bacterium control group）Again in each hole（Except bacterium control group）It is slowly added to final concentration of 50 μ g/mL's
Enzyme solution is incubated 2h in 25 °C, and after the completion of incubation, 3000rpm centrifuges 10min, and enzyme solution is discarded（Except enzyme control group）It is added 8
The SH of concentration various concentration（1024,512,256,128,64, the μ g/mL of 32L, 16 and 8）24 h are incubated in insulating box, finally in every
50 μ L of XTT solution are added in a hole, are protected from light 37 DEG C of incubation, absorbance value of the detection at OD492 nm after 2h.
Comparative example is Proteinase K, aminohexose enzyme, uronic acid enzyme, bacterium control group and enzyme control group.
3, testing result such as Fig. 1 ~ 7.
Fig. 1, various concentration SH in the OD492 of 50 μ g/mL couple 5314 of enzyme L4025 concentration, * p<0.05, with enzyme to photograph
Than after lywallzyme acts on 2h, in 64-1024 μ g/mL, Microflora is remarkably decreased SH concentration with respect to enzyme control group.
Fig. 2, various concentration SH in the OD492 of 50 μ g/mL couple 5314 of RNA enzyme concentration, * p<0.05, with enzyme to photograph
Than after RNA enzyme acts on 2h, in 512-1024 μ g/mL, Microflora is remarkably decreased SH concentration with respect to enzyme control group.
Fig. 3, various concentration SH in the OD492 of 50 μ g/mL couple 5314 of DNA enzymatic concentration, * p<0.05, with enzyme to photograph
Than after DNA enzymatic acts on 2h, only in 512 μ g/mL, Microflora is remarkably decreased SH concentration with respect to enzyme control group.
Fig. 4, comparative example：OD492s, * p of the SH of various concentration in 50ug/ml pairs 5314 of proteinase K concentration<0.05, with
Enzyme control is compared, and Proteinase K is a kind of serine protease that the cleavage activity separated from Candida albicans is wider, it cuts
The carboxy-terminal peptide bond of aliphatic amino acid and aromatic amino acid is cut, main function is the histone that enzymolysis is combined with nucleic acid, is made
DNA is drifted in solution.After Proteinase K acts on 2h, SH concentration does not find Microflora with respect to enzyme within the scope of 8-1024 μ g/mL
The case where control group is remarkably decreased.
Fig. 5, various concentration SH XIV pairs 5314 of protease OD492, * p<0.05, compared with enzyme compares, protease
After XIV acts on 2h, in 128-512 μ g/mL, Microflora is remarkably decreased SH concentration with respect to enzyme control group.
Fig. 6, comparative example：The SH of various concentration is in aminohexose enzyme effect in 5314 OD492, * p<0.05, with enzyme pair
It takes a picture ratio, after aminohexose enzyme effect 2h, SH concentration does not find Microflora with respect to enzyme control group within the scope of 8-1024 μ g/mL
The case where being remarkably decreased.
Fig. 7, comparative example：The SH of the various concentration OD492 under uronic acid enzyme effect to 5314, * p<0.05, with enzyme pair
It takes a picture ratio, after uronic acid enzyme effect 2h, SH concentration does not find that Microflora goes out with respect to enzyme control group within the scope of 8-1024 μ g/mL
The case where being now remarkably decreased.
In all 7 used kind enzymes, the effect of lywallzyme is best, and bacterium can be significantly inhibited in the μ g/ml of SH >=64
Growth illustrates that after lywallzyme destroys candida albicans by membrane matrix, SH further can effectively inhibit bacterium vigor；Protease
Secondly the effect of XIV, bacterium growth can be significantly inhibited in the μ g/ml of SH >=128；The effect of DNA enzymatic and RNA enzyme third, this
When the μ g/ml of SH >=512 when can just significantly inhibit bacterium growth；And the effect of Proteinase K, aminohexose enzyme and uronic acid enzyme is most
It is low, within the scope of SH=8-1024 μ g/ml, significantly inhibiting for bacterium growth is not observed, illustrates the envelope that this 3 kinds of enzymes are digested
Matrix components will not inhibit the antibacterial action of SH.Therefore, pass through the target spot of enzyme directionally hydrolyzing in this experiment, it has been found that lywallzyme
The target spot of hydrolysis may be the most effective target spot that SH plays antimycotic biofilm effect.
Embodiment 9：A method of dissolving candida albicans biofilm extracellular matrix
A method of enzyme solution is added into bacterium solution for dissolving candida albicans biofilm extracellular matrix, and then 25 DEG C of reactions 1.5 are small
When, reaction after the completion of centrifugation enzyme solution to be discarded, then be added decanoy acetaldehyde sodium solution, 37 DEG C reaction 28 hours after XTT is added
Solution reacts 2.5 hours then under the conditions of being protected from light in 37 DEG C；The bacterium solution is to generate the candida albicans for having biofilm
Bacterium solution.10min is centrifuged with 3000rpm when centrifugation.
Preferred embodiment is：A concentration of 50 μ g/mL of the enzyme solution.
Preferred embodiment is：The enzyme of the enzyme solution be selected from lywallzyme, ribonuclease A, deoxyribonuclease I,
Protease XIV.
Preferred embodiment is：A concentration of 700 μ g/mL of the decanoy acetaldehyde sodium solution.
Preferred embodiment is：The XTT solution is by 3,3'- [1- (phenylamino acyl group) -3,4- tetrazoles]-two (4-
Methoxyl group -6- nitros) benzene sulfonic acid sodium salt is dissolved in ringer's solution and obtains.
Embodiment 10：A method of dissolving candida albicans biofilm extracellular matrix
A method of enzyme solution is added into bacterium solution for dissolving candida albicans biofilm extracellular matrix, and then 25 DEG C of reactions 2.5 are small
When, reaction after the completion of centrifugation enzyme solution to be discarded, then be added decanoy acetaldehyde sodium solution, 37 DEG C reaction 20 hours after XTT is added
Solution reacts 1.5 hours then under the conditions of being protected from light in 37 DEG C；The bacterium solution is to generate the candida albicans for having biofilm
Bacterium solution.
Preferred embodiment is：A concentration of 50 μ g/mL of the enzyme solution.
Preferred embodiment is：The enzyme of the enzyme solution be selected from lywallzyme, ribonuclease A, deoxyribonuclease I,
Protease XIV.
Preferred embodiment is：A concentration of 512 ~ 1024 μ g/mL of the decanoy acetaldehyde sodium solution.
Preferred embodiment is：The XTT solution is by 3,3'- [1- (phenylamino acyl group) -3,4- tetrazoles]-two (4-
Methoxyl group -6- nitros) benzene sulfonic acid sodium salt is dissolved in ringer's solution and obtains.
The above-described embodiments merely illustrate the principles and effects of the present invention, and is not intended to limit the present invention.It is any ripe
The personage for knowing this technology can all carry out modifications and changes to above-described embodiment without violating the spirit and scope of the present invention.Cause
This, institute is complete without departing from the spirit and technical ideas disclosed in the present invention by those of ordinary skill in the art such as
At all equivalent modifications or change, should by the present invention claim be covered.
Claims (5)
1. a kind of method of dissolving candida albicans biofilm extracellular matrix, it is characterised in that：Enzyme solution is added into bacterium solution, then
25 DEG C are reacted 1.5 ~ 2.5 hours, are centrifuged after the completion of reaction to discard enzyme solution, and decanoy acetaldehyde sodium solution is then added, and 37 DEG C anti-
XTT solution is added after answering 20 ~ 28 hours, then under the conditions of being protected from light, is reacted 1.5 ~ 2.5 hours in 37 DEG C；The bacterium solution is made a living
At the bacterium solution for the candida albicans for having biofilm.
2. the method for dissolving candida albicans biofilm extracellular matrix according to claim 1, it is characterised in that：The enzyme
A concentration of 50 μ g/mL of liquid.
3. the method for dissolving candida albicans biofilm extracellular matrix according to claim 1, it is characterised in that：The enzyme
The enzyme of liquid is selected from lywallzyme, ribonuclease A, deoxyribonuclease I, protease XIV.
4. the method for dissolving candida albicans biofilm extracellular matrix according to claim 1, it is characterised in that：The fish
A concentration of 512 ~ 1024 μ g/mL of the plain sodium solution of raw meat grass.
5. the method for dissolving candida albicans biofilm extracellular matrix according to claim 1, it is characterised in that：It is described
XTT solution is that-two (4- methoxyl group -6- nitros) benzene sulfonic acid sodium salts of 3,3'- [1- (phenylamino acyl group) -3,4- tetrazoles] are dissolved in woods
Grignard solution and obtain.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101961328A (en) * | 2010-09-17 | 2011-02-02 | 中国人民解放军第二军医大学 | Application of sodium houttuyfonate as synergist of antifungal medicament |
| CN102387794A (en) * | 2009-03-31 | 2012-03-21 | 诺瓦生命科学有限公司 | Inhibition of biofilm organisms |
| CN103690543A (en) * | 2013-12-24 | 2014-04-02 | 广西医科大学 | Composition and method for killing aspergillus fumigatus |
| CN106621824A (en) * | 2017-01-03 | 2017-05-10 | 东北电力大学 | Disintegrant for relieving bio-membrane contamination in membrane bioreactor |
| WO2017197280A1 (en) * | 2016-05-12 | 2017-11-16 | The Trustees Of The University Of Pennsylvania | Compositions and methods for inhibiting biofilm deposition and production |
-
2018
- 2018-05-25 CN CN201810545746.9A patent/CN108753632A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102387794A (en) * | 2009-03-31 | 2012-03-21 | 诺瓦生命科学有限公司 | Inhibition of biofilm organisms |
| CN101961328A (en) * | 2010-09-17 | 2011-02-02 | 中国人民解放军第二军医大学 | Application of sodium houttuyfonate as synergist of antifungal medicament |
| CN103690543A (en) * | 2013-12-24 | 2014-04-02 | 广西医科大学 | Composition and method for killing aspergillus fumigatus |
| WO2017197280A1 (en) * | 2016-05-12 | 2017-11-16 | The Trustees Of The University Of Pennsylvania | Compositions and methods for inhibiting biofilm deposition and production |
| CN106621824A (en) * | 2017-01-03 | 2017-05-10 | 东北电力大学 | Disintegrant for relieving bio-membrane contamination in membrane bioreactor |
Non-Patent Citations (2)
| Title |
|---|
| 吴海芬等: "鱼腥草素抗菌活性的实验研究", 《中国中医药科技》 * |
| 程训民等: "鱼腥草素钠(粉针剂)体外抗菌活性的初步研究", 《时珍国医国药》 * |
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Application publication date: 20181106 |