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Methods For Preventing, Removing, Reducing, or Disrupting Biofilm

a biofilm and reducing technology, applied in the direction of hollow article cleaning, biological water/sewage treatment, compounding agent, etc., can solve the problems of reducing the life of materials, pathogenic problems in municipal water supplies, food processing, etc., and achieve the effect of preventing, reducing, or disrupting the biofilm on the surfa

Inactive Publication Date: 2008-10-09
NOVOZYMES AS +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0012]The term “effective amount” is defined herein as the amount of one or more alpha-amylases that is sufficient to degrade a microbially-produced biofilm comprising alpha-1,4 glucosidic linkages. The effective amount of the one or more alpha-amylase will depend on factors including: the alpha-amylase(s) in question, whether the aim is preventing, removing, or reducing biofilms present on a surface, the period of time desirable for, e.g., degrading a microbially-produced biofilm. High amounts / concentrations of enzyme(s) will in general require shorter times of treatment, while low amounts / concentrations longer times. Further, for instance, preventing biofilm on a surface prone to biofilm formation will in general require lower amounts / concentrations of enzyme(s) than the actual removal of biofilm from a corresponding contaminated surface. However, typical effective usage levels are between 0.005 to 500 mg of alpha-amylase protein per L biofilm control solution, preferably between 0.01-100 mg of enzyme protein per L biofilm control solution. The term “biofilm control solution” refers to a solution used according to the invention for preventing, removing, reducing or disrupting biofilm present on a surface. The method of the invention may result in 10-108-fold, preferably 103-106-fold biofilm reduction in terms of average plate count under the conditions indicated in Example 4 below.

Problems solved by technology

These formations can play a role in restricting or entirely blocking flow in plumbing systems, decreasing heat transfer in heat exchangers, or causing pathogenic problems in municipal water supplies, food processing, medical devices (e.g., catheters, orthopedic devices, implants, endoscopes).
Moreover, biofilms often decrease the life of materials through corrosive action mediated by the embedded microorganisms.
This biological fouling is a serious economic problem in industrial water process systems, pulp and paper production processes, cooling water systems, injection wells for oil recovery, cooling towers, porous media (sand and soil), marine environments, and air conditioning systems, and any closed water recirculation system.
Biofilms are also a severe problem in medical science and industry causing dental plaque, infections (Costerton et al., 1999, Science 284:1318-1322), contaminated endoscopes and contact lenses, prosthetic device colonisation and biofilm formation on medical implants.
As mentioned, biofilm formation causes industrial, environmental and medical problems and the difficulties in cleaning and disinfection of bacterial biofilm with chemicals is a major concern in many industries.
Furthermore, the trend towards milder disinfection and cleaning compositions to reduce their environmental impact may increase the insufficient cleaning of surfaces covered with biofilm.

Method used

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  • Methods For Preventing, Removing, Reducing, or Disrupting Biofilm
  • Methods For Preventing, Removing, Reducing, or Disrupting Biofilm

Examples

Experimental program
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Effect test

example 1

[0108]Biofilm Removal Using Alpha-Amylase A and Al pha-Amylase C

[0109]Biofilm reactors consisted of a 400 ml beaker, a magnetic stirrer, and 2 stainless steel coupons. The coupons are taped vertically to the sides of the beaker so that the bottom edge of the coupon rested on the bottom of the beaker. A stir bar is added and the beakers are covered with a circle of aluminum foil and autoclaved. 200 ml of sterile biofilm medium is added to each beaker. To prepare the inoculum, each bacterial strain (from Bacillus subtilis) is grown overnight at 28° C. on plate count agar. Using a sterile swab, each is suspended in sterile water to an OD686 of 0.100 and then diluted additionally to 10−1. Each assay consisted of 4 control beakers without enzyme, 2 beakers with 50 mg of enzyme protein per liter of solution, and 2 beakers with 100 mg of enzyme protein per liter of solution. Beakers are first incubated at 37° C. overnight with stirring to grow the biofilm on the stainless steel coupons. Fo...

example 2

Raw Starch Solubilization Using Alpha-Amylase A, B, and C

[0110]The rate at which various alpha-amylases solubilize raw, unhydrated wheat starch was measured. Alpha-Amylase A, B and C, respectively, were used in the study.

[0111]Twenty five milliliters of a 1% raw wheat starch solution with pH 8 tris buffer and 15° dH was poured into a tube with lid and placed in a 40° C. water bath. The starting level of “reducing ends” was measured prior to addition of enzyme. The enzyme concentration used in the study was 3 mg enzyme protein per g raw wheat starch. One milliliter samples were taken out at different times. Twenty microliters of 1 M HCl was added prior to incubation at 99° C. for 10 minutes. The combination of acid and heat inactivates the amylase. Then 20 microL 1M NaOH was added to make sure the sample was no longer acidic. The sample was then diluted, incubated with color reagent (PHABH, potassium sodium tartrate, NaOH) at 95° C. for 10 minutes and finally centrifuged before measu...

example 3

[0113]Biofilm Removal Using Alpha-Amylase A and C in Combination with Protease E and Detergent

[0114]A mono-component biofilm of Escherichia coli (ATCC #11229) is grown on polycarbonate coupons in a pre-sterilized CDC biofilm reactor. At the start of the experiment, cultures of E. coli were grown on tryptic soy agar (TSA) overnight at 37° C. The next morning, a single colony was picked from the plate using a 1 microL sterile inoculation loop and added to a solution of 40 g TSB / liter of water. This solution was incubated at 37° C. overnight to grow up the culture. The following day, 1 milliliter of this culture was added to 400 milliliters of minimal media (0.30 g TSB / liter sterile water) contained in the CDC biofilm reactor. The solution was slowly stirred at 130 rpm and grown for 2 days at 22° C. in a non fed batch mode. After the 2 day growth period, the coupon holder rods and coupons were removed from the reactor, rinsed in sterile dilution water to remove planktonic cells, the co...

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Abstract

The present invention relates to methods for preventing, removing, reducing, or disrupting biofilm present on a surface, comprising contacting the surface with an alpha-amylase derived from a bacterium.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application claims priority from U.S. provisional application entitled “Methods for preventing, removing, reducing, or disrupting biofilm”, filed on Sep. 8, 2004 (Ser. No. 60 / 608,535) which is hereby incorporated by reference.FIELD OF THE INVENTION[0002]The present invention relates to improved methods of preventing, removing, reducing, or disrupting biofilm formation on a surface.DESCRIPTION OF THE RELATED ART[0003]Biofilms are biological films that develop and persist at the surfaces of biotic or abiotic objects in aqueous environments from the adsorption of microbial cells onto the solid surfaces. This adsorption can provide a competitive advantage for the microorganisms since they can reproduce, are accessible to a wider variety of nutrients and oxygen conditions, are not washed away, and are less sensitive to antimicrobial agents. The formation of the biofilm is also accompanied by the production of exo-polymeric materials (poly...

Claims

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Application Information

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IPC IPC(8): D06M16/00
CPCB08B7/00B08B9/032B08B17/02C02F3/34C02F3/342C02F2303/20C02F2305/04C11D3/38618C11D11/0023C12N9/2417C12Y304/21062C12Y304/22002C12Y304/22004C12Y304/2404C11D2111/14C11D3/386
Inventor DEINHAMMER, RANDYANDERSEN, CARSTEN
Owner NOVOZYMES AS
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