CN113025447B - Composition for removing bacterial biofilm and application thereof - Google Patents
Composition for removing bacterial biofilm and application thereof Download PDFInfo
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- CN113025447B CN113025447B CN202110164147.4A CN202110164147A CN113025447B CN 113025447 B CN113025447 B CN 113025447B CN 202110164147 A CN202110164147 A CN 202110164147A CN 113025447 B CN113025447 B CN 113025447B
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- 238000004321 preservation Methods 0.000 claims description 4
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- 238000012360 testing method Methods 0.000 abstract description 11
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- 230000003213 activating effect Effects 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical class N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
- C11D3/16—Organic compounds
- C11D3/38—Products with no well-defined composition, e.g. natural products
- C11D3/386—Preparations containing enzymes, e.g. protease or amylase
- C11D3/38636—Preparations containing enzymes, e.g. protease or amylase containing enzymes other than protease, amylase, lipase, cellulase, oxidase or reductase
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
- A01N37/44—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing at least one carboxylic group or a thio analogue, or a derivative thereof, and a nitrogen atom attached to the same carbon skeleton by a single or double bond, this nitrogen atom not being a member of a derivative or of a thio analogue of a carboxylic group, e.g. amino-carboxylic acids
- A01N37/46—N-acyl derivatives
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/10—Animals; Substances produced thereby or obtained therefrom
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/50—Isolated enzymes; Isolated proteins
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- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
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- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D7/00—Compositions of detergents based essentially on non-surface-active compounds
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- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D7/00—Compositions of detergents based essentially on non-surface-active compounds
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- C11D7/46—Animal products
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- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D2111/00—Cleaning compositions characterised by the objects to be cleaned; Cleaning compositions characterised by non-standard cleaning or washing processes
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Abstract
The invention discloses a composition for removing bacterial biofilm and application thereof. The composition for removing the bacterial biofilm comprises dextranase, an anti-dextran monoclonal antibody and a buffer solution. The detection test result of the trace bacterial envelope of the invention shows that: when the dextranase with the concentration of 0.2-0.3U/mL and the anti-dextran monoclonal antibody D24 with the concentration of 1-3 mg/mL are added in combination, the clearance rate of the anti-dextran monoclonal antibody to the leuconostoc mesenteroides biofilm is 32.7% -69.6%; compared with the single addition, the combination addition of the dextranase and the anti-dextran monoclonal antibody D24 has obvious synergistic effect on the clearance rate of the leuconostoc mesenteroides biofilm. Therefore, the composition for removing the bacterial biofilm can effectively remove the bacterial biofilm, and particularly can effectively remove the bacterial biofilm on the surface of equipment in the sugar industry.
Description
Technical Field
The invention belongs to the field of sugar production, and particularly relates to a composition for removing a bacterial biofilm and application thereof.
Background
In the process of sugar production (including sugar production by sugarcane and sugar production by beet), juice extraction is the first process of sugar production. The juice extraction process is to extract the juice rich in cane sugar from sugar crops by means of squeezing or exudation and the like. The sugar material is typically harvested and stored in the field or at a point of stacking for a period of time before being extracted. The sugar material loses immunity after being harvested, a large number of cuts or burnt wounds exist, juice rich in nutrition in the sugar material is exposed, and soil containing a large number of microorganisms and the like are mixed in the harvesting process, so that the microorganisms are rapidly propagated. When juice extraction is carried out, sugar materials contain a large amount of microorganisms, a workshop lacks means for killing microorganisms and the production environment is very suitable for the growth of the microorganisms, so that the microorganisms are further propagated. Due to the large amount of microorganisms and nutrients existing for a long time, bacterial biofilms are formed on the surfaces of the equipment and the pipelines, so that the microorganisms are fixed on the surfaces of the equipment and the pipelines, and are propagated by continuously utilizing the nutrients like a catalytic center, thereby causing the loss of cane sugar.
Bacterial biofilms are widely present on a variety of aqueous moist surfaces and are a structural bacterial community consisting of bacterial cells attached to the surface of an inert or active entity and a hydrated matrix encasing the bacteria, typically composed of multiple species. The bacteria account for less than 1/3 in the biofilm, and the rest is sticky substances and extracellular polysaccharide secreted by the bacteria, which is the key point for the bacterial biofilm to be adhered to the surface of an object.
Leuconostoc mesenteroides is a common bacterium in the sugar production process, is usually present in air, soil, plant body surface and the like, and can ferment sucrose to generate high-viscosity dextran. Dextran is an important component constituting bacterial biofilms in sugar manufacturing facilities, and mainly plays a role in causing microorganisms to adhere to object surfaces or to microorganisms. Therefore, Leuconostoc mesenteroides and the dextran secreted by the Leuconostoc mesenteroides are important factors for forming bacterial biofilm on the surface of sugar manufacturing equipment.
At present, in actual production, workshop staff only wash sugar making equipment with water flow for cleaning, and bacterial biofilms cannot be effectively removed.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides a composition for removing bacterial biofilm and application thereof.
The first object of the present invention is to provide a composition for removing bacterial biofilm, comprising a) dextranase, b) an anti-dextran monoclonal antibody, and c) a buffer.
Preferably, the concentration of the dextranase is 0.1-0.3U/mL.
Preferably, the concentration of the anti-dextran monoclonal antibody is 1-3 mg/mL.
Preferably, the concentration of the dextranase is 0.2-0.3U/mL, and the concentration of the anti-dextran monoclonal antibody is 1-3 mg/mL. Further preferably, the concentration of the dextranase is 0.3U/mL, and the concentration of the anti-dextran monoclonal antibody is 2 mg/mL.
Preferably, the buffer is phosphate buffer. More preferably, the buffer is 0.2mol/L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer with pH 6.5.
Preferably, the anti-dextran monoclonal antibody is an anti-dextran monoclonal antibody D24, and the anti-dextran monoclonal antibody D24 is generated by a dextran hybridoma cell strain D24 with the preservation number of CGMCC No. 21002.
The second purpose of the invention is to provide the application of the composition for removing the bacterial biofilm in the removal of the bacterial biofilm.
Preferably, the bacterial biofilm is a bacterial biofilm on the surface of equipment in the sugar industry.
Preferably, the bacterial biofilm is a leuconostoc mesenteroides biofilm.
The detection test result of the trace bacterial envelope of the invention shows that: when the dextranase with the concentration of 0.1-0.3U/mL is independently added, the clearance rate of the dextranase to the leuconostoc mesenteroides biofilm is 10.1-28.3%; when the anti-dextran monoclonal antibody D24 with the concentration of 1-3 mg/mL is independently added, the anti-dextran monoclonal antibody has almost no clearing effect on the leuconostoc mesenteroides biofilm; when the dextranase with the concentration of 0.2-0.3U/mL and the anti-dextran monoclonal antibody D24 with the concentration of 1-3 mg/mL are added in combination, the clearance rate of the anti-dextran monoclonal antibody to the leuconostoc mesenteroides biofilm is 32.7% -69.6%. Compared with the single addition, the combination addition of the dextranase and the anti-dextran monoclonal antibody D24 has obvious synergistic effect on the clearance rate of the leuconostoc mesenteroides biofilm. Therefore, the composition for removing the bacterial biofilm can effectively remove the bacterial biofilm, and particularly can effectively remove the bacterial biofilm on the surface of equipment in the sugar industry.
The glucan hybridoma cell strain D24 is preserved in China general microbiological culture Collection center (CGMCC) in 2020, 10 months and 16 days, and has the following address: west road No. 1, north west of the morning area, beijing, 3, institute for microbiology, china academy of sciences, accession number: CGMCC No: 21002.
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto.
Example 1: composition for removing biofilm of Leuconostoc mesenteroides
1. Reagent and strain for experiment
Dextran enzyme: purchased from Zhongnuo biotechnological development Jiangsu Co.
Anti-dextran monoclonal antibody D24: produced by a glucan hybridoma cell strain D24 with the preservation number of CGMCC No. 21002.
Disodium hydrogenphosphate-sodium dihydrogenphosphate buffer (0.2mol/L, pH 6.5): purchased from Shanghai Pongjing industries, Ltd.
Leuconostoc mesenteroides: purchased from Guangdong province culture Collection of microorganisms, and having GIM number 1.473.
Liquid culture medium: 130.0g of sucrose, 2.0g of peptone and KH2PO4 0.3g、Na2HPO41.4g, adding water to dissolve, diluting to 1L, and sterilizing at 121 deg.C for 20 min.
Solid medium: 130.0g of sucrose, 2.0g of peptone and KH2PO4 0.3g、Na2HPO41.4g of agar and 15g of agar are dissolved in water, the volume is determined to be 1L, the mixture is sterilized for 20min at 121 ℃, poured into a culture dish while the mixture is hot and cooled for standby.
2. Preparation of anti-dextran monoclonal antibody D24
The method comprises the steps of inoculating 0.5mL of liquid paraffin into the abdominal cavity of a 6-8-week-old BALB/c mouse (purchased from southern medical university laboratory animal center) with strong physique and good growth condition, inoculating 5 x 10 dextran hybridoma cell strain D24 (with the preservation number of CGMCC No.21002) diluted by a serum-free culture medium (purchased from Thermo Fisher scientific) into the abdominal cavity after two weeks, and inoculating 5 x 10 dextran hybridoma cell strain D24 into the abdominal cavity of the mouse50.2 mL. After 8 days, observing the ascites generation condition of the mice every day, when the ascites is as much as possible and the mice are dying, killing the mice, and sucking the ascites out under the aseptic condition. Standing at room temperature for 30min, centrifuging at 10000rpm for 10min, collecting supernatant to obtain pretreated ascites, and freezing at-70 deg.C.
The purification scheme is an octanoic acid-ammonium sulfate precipitation method. Adding 2 parts of 0.06mol/L acetic acid buffer solution with pH4.0 into 1 part of pretreated ascites, and adjusting the pH to 4.5 by using 1mol/L HCl; adding 11 mu L of octanoic acid into diluted ascites per ml, dropwise adding octanoic acid under stirring at room temperature within 30min, standing at 4 deg.C for 2 hr, centrifuging at 12000rpm for 30min, and removing precipitate; filtering the supernatant with a nylon sieve (the diameter of the nylon sieve is 125 μm), adding 1/10 volumes of 0.1mol/L PBS buffer solution with pH7.2, and adjusting the pH to 7.2 with 1mol/L NaOH; adding saturated ammonium sulfate at 4 deg.C to 45% saturation, mixing gently for 30min, and standing for 1 hr; centrifuging at 12000rpm for 30min, and discarding the supernatant; dissolving the precipitate in 50-100 times volume of PBS buffer solution, dialyzing with 50-100 times volume of PBS, and standing overnight at 4 ℃; taking out the mixture and centrifuging the mixture for 30min at 12000rpm, removing insoluble precipitates, and freeze-drying the mixture to obtain freeze-dried powder which is named as anti-dextran monoclonal antibody D24 freeze-dried powder for later use.
3. Preparation of the composition: according to the mixture ratio of dextranase and anti-dextran monoclonal antibody D24 in the composition corresponding to test groups 1-16 in the following table 1, dextranase and anti-dextran monoclonal antibody D24 (the anti-dextran monoclonal antibody D24 freeze-dried powder prepared in step 2) are dissolved in 0.2mol/L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution with the pH value of 6.5.
4. Detection test for trace amount of bacterial envelope
The removal degree of the composition on the leuconostoc mesenteroides biofilm is detected by a trace bacterial envelope detection test (a 96-well plate biofilm formation test).
The specific test method is as follows: dissolving Leuconostoc mesenteroides freeze-dried powder into a liquid culture medium, culturing for 24 hours at 37 ℃, then, scribing and activating on a solid culture medium, and culturing for 48 hours at 37 ℃; picking a single colony, inoculating the single colony in 50mL of liquid culture medium, and culturing at 37 ℃ for 48 hours to obtain a culture solution; thirdly, diluting the culture solution by 200 times with a liquid culture medium, inoculating the diluted culture solution to a 96-hole plate polystyrene microplate, keeping the volume of each well at 200 mu L, and standing and culturing at 37 ℃ for 48 hours; fourthly, abandoning the bacterial liquid, washing the plate for 3 times by PBS, and drying the plate; adding the composition with each concentration (the components are shown in the following table 1 specifically), allowing the mixture to act at normal temperature for 15min, wherein each pore is 200 mu L; sixthly, the solution is discarded, and the plate is washed for 3 times by PBS and dried; seventhly, adding 200 mu L of 2 percent crystal violet solution into each hole, and dyeing for 5min at room temperature; eighthly, discarding the dye solution, washing the plate with water, and airing; add 100. mu.L of 33% acetic acid solution to each well and stand at 37 ℃ for 30 min; ninthly, measuring an OD value at 590nm (zero adjustment is carried out by using a 33% acetic acid solution) by using a microplate reader, and reading the OD value of each test group in three-hole mode; the clearance rate of the composition on the leuconostoc mesenteroides biofilm is calculated by the following formula in the R: (OD value of test group 1-OD value of this group) ÷ OD value of test group 1. times.100%.
TABLE 1 compositions of test groups 1-16 and clearance of Leuconostoc mesenteroides biofilm
Note: through significance test (t test, letter marking method), the same letter exists among the mean values, the difference is not significant, and the difference is not significant if the same letter does not exist.
As can be seen from the above table 1, when the concentration of the separately added dextranase is 0.1-0.3U/mL, the clearance rate of the dextranase to the leuconostoc mesenteroides biofilm is 10.1-28.3%; when the concentration of the anti-dextran monoclonal antibody D24 added alone is 1-3 mg/mL, the anti-dextran monoclonal antibody has almost no effect of clearing leuconostoc mesenteroides biofilm; when the dextran enzyme concentration is 0.2-0.3U/mL and the anti-dextran monoclonal antibody D24 concentration is 1-3 mg/mL, the clearance rate to the leuconostoc mesenteroides biofilm is 32.7% -69.6%. Compared with the method of adding the dextranase and the anti-dextran monoclonal antibody D24 independently, the combination of the dextranase and the anti-dextran monoclonal antibody D24 has obvious synergistic effect on the clearance rate of the leuconostoc mesenteroides biofilm. Therefore, the combination of dextranase and anti-dextran monoclonal antibody D24 can effectively remove leuconostoc mesenteroides biofilm, especially effectively remove bacterial biofilm on the surface of equipment in sugar industry.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Claims (7)
1. A composition for removing a bacterial biofilm comprising a) dextranase, b) an anti-dextran monoclonal antibody, and c) a buffer; the concentration of the dextranase is 0.1-0.3U/mL; the concentration of the anti-dextran monoclonal antibody is 1-3 mg/mL; the anti-dextran monoclonal antibody is an anti-dextran monoclonal antibody D24, and the anti-dextran monoclonal antibody D24 is generated by a dextran hybridoma cell strain D24 with the preservation number of CGMCC No. 21002.
2. The composition for removing a bacterial biofilm according to claim 1, wherein the concentration of said dextranase is 0.2-0.3U/mL, and the concentration of said anti-dextran monoclonal antibody is 1-3 mg/mL.
3. A bacterial biofilm removing composition according to claim 1, wherein said buffer is a phosphate buffer.
4. A bacterial biofilm removing composition according to claim 3, wherein said buffer is 0.2mol/L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer at pH 6.5.
5. Use of a bacterial biofilm removing composition as claimed in any one of claims 1 to 4 for removing bacterial biofilms.
6. The use according to claim 5, wherein the bacterial biofilm is a bacterial biofilm on equipment in the sugar industry.
7. The use of claim 5, wherein the bacterial biofilm is a Leuconostoc mesenteroides biofilm.
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Address after: No.10, shiliugang Road, Haizhu District, Guangzhou City, Guangdong Province 510000 Patentee after: Institute of biological and medical engineering, Guangdong Academy of Sciences Address before: No.10, shiliugang Road, Haizhu District, Guangzhou City, Guangdong Province 510000 Patentee before: Institute of bioengineering, Guangdong Academy of Sciences |