WO2000059834A1

WO2000059834A1 - Methods for inhibiting the production of slime in aqueous systems

Info

Publication number: WO2000059834A1
Application number: PCT/US2000/006435
Authority: WO
Inventors: Roy Hernandez-Mena; James A. Romesser
Original assignee: Betzdearborn Inc.
Priority date: 1999-04-02
Filing date: 2000-03-29
Publication date: 2000-10-12
Also published as: AU4008800A

Abstract

Methods for inhibiting the attachment of microbes to surfaces in aqueous systems are disclosed. Flavanol compounds are added to aqueous systems, such as papermaking and cooling water systems, to inhibit slime formation problems caused by microbial attachment to surfaces.

Description

METHODS FOR INHIBITING THE PRODUCTION OF SLIME IN AQUEOUS SYSTEMS

FIELD OF THE INVENTION The present invention relates to methods for inhibiting the formation of slime by microorganisms in industrial aqueous systems. More particularly, these methods are directed towards inhibiting the attachment of microorganisms to the surfaces of industrial aqueous systems.

BACKGROUND OF THE INVENTION Microorganisms and the slimes they produce are responsible for the formation of deposits in papermaking and industrial cooling water systems. Bacterial slimes are composed of exopolysaccharides (EPS) which exist as capsules or slime layers outside of the cell walls. When these slimes form on surfaces in paper or cooling systems, they trap organic and inorganic components and debris present in the process waters. As the microorganisms grow within paper system deposits, portions of the deposit may detach from the surface and cause paper breaks and spots in produced paper, which reduces the paper quality and increases machine downtime. Microbial growth and slime formation in cooling systems results in reduced heat exchange and plugging of heat exchanger tubes, excessive fouling of the cooling water, tower decks and fill, and is a potential cause of under-deposit corrosion. The term "slime" is a broad one covering a wide range of viscous, mucous, or leathery materials and mixtures found in industrial waters. Slimes are polymeric in nature and can be broadly classified as chemical, biological, or composite slimes depending upon their cause or composition. For example, raw materials and equipment used in the paper industry are not sterile and water used in conjunction with such equipment is continuously being contaminated with a wide variety of microorganisms from such sources as wood pulp, chemicals, air, makeup water, and the like. The growth of certain specific forms of these biological contaminants causes or produces polymeric excretions or products that are or become slime.

Historically, slime formation has been treated by the addition to industrial waters (e.g., white water associated with the pulp and paper industry) of slimicides. The purpose of these slimicides is to destroy or arrest the growth of some of the many organisms present in the water to thereby prevent or retard the formation of slime. Chemicals used as slimicides have included chlorine, phenylmercuric acetate, pentachlorophenol, tributyl tin oxide, and isothiocyanates, all of which are relatively toxic to humans. Microbially produced exopolysaccharides can build up, retard heat transfer and restrict water flow through cooling water systems. Controlling slime-forming bacteria by applying toxic chemicals is becoming increasingly unaccepted due to environmental problems. In addition, the efficacy of the toxicants is minimized by the slime itself, since the extracellular polysaccharide surrounding microorganisms impedes toxicant penetration.

Toxicants cannot adequately control large populations of attached bacteria, and they are effective mainly against suspended microorganisms. Although surfactants and dispersants which penetrate and help loosen slime can enhance the activity of toxicants, they are nonspecific and may have deleterious effects on the industrial process or the environment. Recently, methods directed at controlling microbial slimes include the use of enzymes.

These approaches attempt to disrupt the attachment process so that slime formation is prevented, or by hydrolyzing the exopolysaccharide (EPS) produced by the microorganisms after attachment. Using an enzyme to control slime will require knowledge of the composition of the slime, so that an appropriate enzyme-substrate combination is employed. SUMMARY OF THE INVENTION

The present invention relates to methods for inhibiting the formation of slime in industrial aqueous systems such as papermaking and cooling water systems. The slime formation is inhibited by preventing the attachment of microorganisms to the surfaces of the aqueous system wherein the slime-producing bacteria are present. It has been found that the addition of flavanol compounds to these aqueous systems inhibits the attachment of microorganisms, particularly bacteria.

DESCRIPTION OF THE RELATED ART U.S. Pat. No. 5,695,652 discloses methods for inhibiting attachment of microbes to surfaces of aqueous systems with tannin and/or tea extracts. The tea extracts employed are those obtained after extraction of teas in various forms with 45% ethanol.

"Oolong Tea Polyphenols Inhibit Experimental Dental Caries in SPF Rats Infected with Mutans Streptococci," T. Ooshima et al., Caries Res 27: 124-129, 1993, discusses the inhibitory effects of Oolong tea extracts derived from Camellia sinensis on dental caries in specific pathogen-free rats. This study indicated that Oolong tea extracts contain polyphenols and inhibit insoluble glucan synthesis by inhibiting glucosyltransferases (GTases) and the sucrose-dependent cell adherence of Streptococcus mutans. The antifouling properties of phenolic acid sulphates isolated from marine organisms is discussed in "The Antifouling Activity of Natural and Synthetic Phenolic Acid Sulphate Esters," J.S. Todd et al., Phytochem 34(2) 401-404, 1993. This study found that p-(sulphooxy) cinnamic acid, isolated from the seagrass Zoster a marina, prevents attachment of marine bacteria and barnacles to artificial surfaces.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to methods for inhibiting the attachment of microorganisms to the surfaces of aqueous systems comprising adding to the aqueous system an effective inhibiting amount of a flavanol compound. The flavanol compounds are typically found as active components in tea fractions.

The flavanol compound generally has the formula:

wherein R, is selected from H and OH, R₂ is selected from H, OH, and 0(3,4,5- trihydroxybenzoyl), and R₃ is selected from H or OH. Representative flavanols include but are not limited to

(-)-epicatechin, (-)-epicatechin gallate, (-)-epigallocatechin,

(-)-epigallocatechin gallate, (+)-catechin, and (+)-gallocatechin.

The compounds employed in the present invention will inhibit the attachment of microbes to surfaces in aqueous systems. By inhibiting the attachment, the formation of slimes will be inhibited. The methods of the present invention are best employed in industrial aqueous systems where microbial slimes are a problem. Systems which are particularly susceptible to slime proliferation are papermaking and cooling water systems where poor paper quality, machine downtime and fouled heat exchangers result from slime formation. The microbes that can be inhibited by the methods of the present invention include but are not limited to Pseudomonas, Klebsiella, Aerobacter, Acinetobacter, Enterobacter, and Flavobacterium.

For purposes of the present invention, the phrase "effective inhibiting amount" is that amount of tea fraction or flavanol compound which is sufficient to inhibit attachment of microbes. This amount will vary according to the conditions of the aqueous system to be treated.

Preferably, the tea fraction or flavanol compound is added to the aqueous system in an amount ranging from about 5 parts to about 500 parts per million parts of the aqueous system. The tea fraction or flavanol compound may be applied to the aqueous phase in contact with the surface experiencing or having the potential to experience microbial fouling. The tea fraction or flavanol compound may also be applied directly to the surface experiencing or having the potential to experience microbial fouling.

The tea fraction or flavanol compound may be applied neat or as a solution. The solvent may be water or alcohol; however, any solvent that is compatible with both the tea fraction or flavanol compound and the aqueous system to be treated may be employed. In addition, the tea fraction or flavanol compound may be added in conjunction with biocides and/or surfactants, as well as corrosion or scale inhibitors, as an adjunct to a complete microbial control program. The invention will now be described with reference to a number of specific examples which are to be regarded solely as illustrative and not as restricting the scope of the invention.

EXAMPLES Dried Orange Pekoe Tea Extract which was prepared by extracting tea leaves with 45% ethanol was initially partitioned into two phases which were generated using ethyl acetate, butanol and water in a ratio of 1 :1 :1. The Upper Non-Polar Phase (#1) of this fractionation was dried and resuspended in ethyl acetate. This material was then repartitioned by solvent/solvent extraction using ethyl acetate, ethyl ether and water in a 1:1:1 ratio. Previous studies indicated that efficacious materials would be present in both phases (Non-Polar Phase #2 and Polar Phase #2) and this was confirmed by the bacterial adhesion assay described below.

To a flask containing 50 ml of a simple salts medium (SSM) and 3 g/L of glucose was added 50 ml of a solution containing 10 μCi/ml of ³H-adenine. This flask was inoculated with a field isolate identified as Pseudomonas aeruginosa. The flask was incubated overnight at 37° C with shaking at 200 rpm. This procedure generated a radio-labeled culture which could be monitored using a scintillation counter.

Following incubation the culture was centrifuged at 8,000 x g for 10 minutes in a Sorvall RC 26 Plus centrifuge. The supernatant was decanted and the culture was re- suspended in SSM to remove any non-incorporated radio-label. Centrifugation was repeated, the supernatant was decanted and the pellet was re-suspended in five ml of SSM. In a side- arm flask containing SSM, approximately 5 to 10 ml, a 300 Klett Unit suspension was prepared by adding an appropriate amount of a cell suspension to the flask. The suspension contained approximately 10⁹ CFU/ml, and was used in the assay described below.

The assay used to test compounds for anti-sessile activity is microplate -based and uses Packard Optiplates. In a typical assay, the wells are pre-wetted with 25 μL of SSM for 30 minutes. The test compound is added to six wells of the microplate at a volume of 50 μl per well. To wells used as controls, 50 μL of sterile de-ionized water is added. This is followed by addition of 50 μL of the radio-labeled cell suspension discussed above. Immediately after inoculation, one row of control wells is harvested to establish the initial amount of attachment to the test surface (T = 0 hr). Harvesting removes any unattached cells and is accomplished by rinsing the wells with de-ionized water using a Skatron Titertek Cell Harvester. Any remaining wash water is removed and the plate is incubated with shaking. At one hour, the remaining wells are harvested as outlined above. The amount of radio-label remaining in treated wells is compared with those used as controls.

The results of this testing are presented in Table I. The values listed in column "% Attachment" represent the amount of ³H-labeled bacteria that attached to the test surface with respect to controls that did not undergo any treatment. The lower the number in this column, the fewer bacteria attached to the surface.

Table I

Bacterial Adhesion Assay using Various Tea Fractions Test Organism: Pseudomonas aeruginosa

Avg. Standard %

Concentration (ppm CPM CPM Deviation Attachment

Control:

5,322 6,397 5,487 6,458 4,110 6,145

5,179 6,241 5,045 5,598 781

Non-polar Phase #2

500 1,013 989 578 860 245 15

250 1,989 2,368 2,260 2,206 195 39

125 2,797 2,167 1,647 2,204 576 39

63 3,503 2,741 3,112 3,1 19 381 56

31 3,686 5,236 4,637 4,520 782 81

16 5,310 5,478 5,473 5,420 96 97

Polar Phase #2

500 1,227 1,249 887 1,121 203 20

250 2,437 2.652 2,385 2,491 142 45

125 1,799 2,151 1,729 1,764 49 32

63 3,146 2,792 3,425 3,121 317 56

31 8,965 5,350 4,347 6,221 2,429 111

16 5,864 5,589 5,512 5,655 185 101

The results of this testing demonstrate that both sets of fractions showed roughly equal performance. Further processing was performed on Non-Polar Phase #2 by Centrifugal

Partition Chromatography (CPC).

The differential partitioning of compounds between two immiscible liquids to extract and purify materials has been used for many years. CPC is a similar technique in that the separation mechanism is based on the difference in distribution of components over two immiscible liquid phases. The general features of CPC have been described by Berthod and

Armstrong (1988). See, Centrifugal Partition Chromatography. I. General Features, Berthod,

Alain and Daniel W. Armstrong, J. Liq Chromatogr, 11(3)547-566(1988).

Using ethyl acetate, ethyl ether and water, a separation was conducted on the Non-Polar Phase

#2 described above. Separation conditions were as follows: Protocol for CPC Fractionation of Non-Polar Phase #2

CPC system: PC, Inc.

UV Detector: Water's Photodiode Array

Wavelength Monitored 270 nm

Flow rate: 3 mL/min

Injection: -200 mg to 950 mg

PDA data: 195-400 nm

Fraction size collected: 9 mL

Solvents: Ethyl Ether:Ethyl Acetate: H₂O, (1 :1 :1)

Run time: 240 minutes

Moblie phase: Upper phase (for first 120 minutes)

Lower phase (for remaining 120 minutes) Fractions collected: 80

Six fractions based on peaks observed in the chromatogram were pooled from the eighty fractions collected over the course of the separation. The pooled fractions were dried and evaluated using the Bacterial Adhesion Assay. The results of this testing are presented in

Table II.

Table II

Bacterial Adhesion Assay of CPC Generated Fractions Test Organism: Pseudomonas aeruginosa

Avg. Standard %

Concentration (ppm) CPM CPM Deviation Attachment

Control:

2,458 2,750 2,489 2,798 2,265 2,729 2,325 2,621 1,532 2,441 389

Non-polar Phase #2

500 166 194 193 184 16 8

250 269 264 278 270 7 11

125 796 844 659 766 96 31

63 1,787 1,834 1,590 1,737 129 71

31 2,346 2,286 2,334 2,322 32 95 Table II. continued

Avg. Standard %

Concentration (pprr CPM CPM Deviation Attachment

Fraction 1 500 136 141 125 134 8 5

250 174 152 221 182 35 7

125 707 727 697 710 15 29

63 1,151 1,191 1,162 1,168 21 48

31 1,868 1,867 1,860 1,865 4 76 Fraction 2

500 503 456 461 473 26 19

250 691 718 761 723 35 30

125 1,240 1,093 1,154 1,162 74 48

63 1,578 1,535 1,722 1,612 98 66 31 2,261 2,381 2,389 2,344 72 96

Fraction 3

500 487 521 494 501 18 21

250 744 674 693 704 36 29

125 1,051 1,074 1,005 1,043 35 43 63 1,746 1,590 1,249 1,528 254 63

31 2,624 2,485 2,479 2,529 82 104

Fraction 4

500 709 698 669 692 21 28 250 794 815 795 801 12 33

125 1,199 1,106 1,181 1,162 49 48

63 1,958 1,835 1,805 1,866 81 76

31 2,667 2,543 2,645 2,618 66 107 Fraction 5 500 340 308 253 300 44 12

250 438 441 365 415 43 17

125 820 749 747 772 42 32

63 1,409 1,311 1,173 1,298 119 53

31 2,487 2,068 2,203 2,253 214 92 Fraction 6

500 447 613 535 532 83 22

250 892 907 874 891 17 37

125 1,387 1,361 1,395 1,381 18 57

63 2,044 2,187 2,250 2,160 106 89 31 2,541 2,676 2,572 2,596 71 106 As demonstrated in Table II, all of the fractions showed activity against bacterial attachment. Analytical HPLC revealed that Fractions 1 and 2 were quite similar compared to the other fractions tested. These were therefore pooled and used in the purification steps described below. In order to reduce the amount of material to be processed by preparatory or semi- preparatory HPLC, the pooled CPC Fractions 1 and 2 were further extracted by first dissolving the material in a minimal amount of ethyl acetate and extracting it with a 2X volume of distilled water. This resulted in about 90% of the material remaining in the ethyl acetate phase. Both phases were dried and stored. The ethyl acetate phase was then redissolved in a minimal amount of distilled water. This solution was then extracted with an equal volume of ethyl ether. The phases were separated and dried. The ethyl ether phase contained about 35.3% of the material. The fractions were tested in the Bacterial Adhesion Assay and the results are presented in Table III.

Table III

Bacterial Adhesion Assay of Solvent/Solvent Extracted CPC Fractions Test Organism: Pseudomonas aeruginosa

Avg. Standard %

Concentration (ppm) CPM CPM Deviation Attachment

Control:

3,794 4,405 3,828 4,084 3,830 4,564

4,004 4.555 4,155 4,135 308

Ethyl Acetate H₂0 Phase

500 582 422 487 497 80 12

250 794 696 671 720 65 17

125 1,687 1,662 1,692 1,680 16 41

63 2,062 2,309 2,066 2,146 141 52

31 3,303 3,141 2,997 3,147 153 76

Ethyl Ether H₂0 Phase

500 99 117 96 104 11 3

250 139 124 117 127 11 3

125 618 547 640 602 49 15

63 1,706 1,362 1,460 1,509 177 36

31 2,090 2,171 2,163 2,141 45 52

Ethyl Ether Phase

500 92 71 97 87 14 2

250 166 145 136 149 15 4

125 1,005 926 824 918 91 22

63 1,553 1,369 1,581 1,501 115 36

31 2,637 2,169 2,661 2,489 277 60 All three phases demonstrated efficacy at inhibiting bacterial adhesion. The ethyl ether extract generated above was further fractionated by semi-preparatory HPLC to yield 35 fractions which were collected over the course of the HPLC run. The protocol for the fractionation was as follows:

Semi-Prep HPLC Fractionation Conditions

Column: Lichroshper, Merck(10 X 250 mm, 100RP₁₈ , , 10 μm)

Flow rate: lO mL/min

Injection: 250 μL

PDA data: 200-600 nm

Solvents: A = 0.1% TFA in H₂O B = Acetonitrile

Time(min): Gradient Conditions:

0 95% A , 5% B

0-30 60% A, 5% B

30-35 100% B

Collection time: 1 min

# of Fractions: 35

Those fractions were then dried and tested using the Bacterial Adhesion Assay. The individual fractions collected did not contain sufficient material to make accurate mass determinations. As a consequence, all fractions were suspended in 1 mL of deionized water for testing purposes.

The results of this testing are presented in Table IV.

Table IV

Bacterial Adhesion Assay of Semi-Preparatory HPLC

Generated Fractions

Test Organism: Pseudomonas aeruginosa

Avg. Standard %

Fraction # CPM CPM Deviation Attachment

Control:

5,999 6,990 6,824 7,003 6,033 6,464

5,713 7,262 5,769 6,451 590

1 5,968 6,666 6,095 6,243 372 97

2 5,583 6,192 5,708 5,828 322 90

3 5,935 6,123 6,081 6,046 99 94

4 6,073 6,847 6,553 6,491 391 101

5 6,042 5,757 6,280 6,026 262 93

6 5,162 5,608 5,629 5,466 264 85

7 6,141 6,042 6,476 6,220 227 96

8 6,224 5,285 5,911 5,807 478 90

9 4,931 5,753 6,067 5,584 587 87

10 3,195 2,525 2,220 2,647 499 41

11 1,291 1,057 1,255 1,201 126 19

12 3,572 3,435 3,316 3,441 128 53

13 4,797 6,234 5,160 5,397 747 84

14 829 971 928 909 73 14

15 1,484 1,694 999 1,392 356 22

16 2,685 2,788 2,734 2,736 52 42

17 3,108 3,169 3,392 3,223 150 50

18 2,441 2,263 2,189 2,298 130 36

19 2,502 2,199 3,329 2,677 585 41

20 3,269 2,928 3,533 3,243 303 50

21 2,689 2,550 2,665 2,635 74 41

22 2,615 2,139 2,194 2,316 260 36

23 2,298 2,402 1,945 2,215 240 34

24 1,100 1,009 1,104 1,071 54 17

25 1,188 1,149 1,265 1,201 59 19

26 2,559 2,732 3,478 2,923 488 45

27 2,187 2,158 3,643 2,663 849 41

28 1,961 1,897 1,326 1,728 350 27

29 5,006 4,732 4,592 4,777 211 74

30 5,073 4,617 5,131 4,940 282 77

31 3,668 3,574 3,863 3,702 147 57

32 3,902 4,245 4,172 4,106 181 64

33 4,191 4,024 4,370 4,195 173 65

34 4,482 3,623 3,868 3,991 443 62

35 5,147 5,287 4,923 5,119 184 79 Of the fractions shown in Table IV, 10, 11, 14, 15, 24, 25 and 28 proved most efficacious. These fractions were further analyzed by analytical HPLC to determine the level of purification. Fractions 11 and 14 were found to be relatively pure and were analyzed by electrospray ionization/mass spectroscopy and nuclear magnetic resonance (NMR) spectroscopy and found to be (-)-epigallocatechin gallate and (-)-epicatechin gallate, respectively.

These two compounds were obtained in pure form from a chemical supplier and tested for their ability to prevent attachment in the bacterial adhesion assay. These results are presented in Table V. Table V

Adhesion Assay to Test Efficacy of (-)-Epigallocatechin gallate and (-)-Epicatechin gallate Test Organism: Pseudomonas aeruginosa

Avg. Standard %

Concentration (ppml CPM CPM Deviation Attachment Control:

4,671 5,304 4,831 5.030 4,874 5,179

5,035 5,323 5,089 5,037 218

(-)-Epigallocatechin gallate

500 635 819 688 714 95 14

250 1,390 1,160 1,256 1,269 116 25

125 1,943 1,929 1,941 1,938 8 38

63 2,383 2,636 2,508 2,509 127 50

31 2,284 3,107 3,118 2,836 478 56

16 3,501 4,480 4,653 4,211 621 84

8 3,057 3,953 4,170 3,727 590 74

4 4,559 4,784 4,594 4,649 127 92

(-)-Epicatechin gallate

500 1,205 1,293 1,266 1,288 20 24

250 1,347 1,646 1,629 1,541 168 31

125 2,767 2,613 2,663 2,681 79 53

63 3,629 3,144 3,349 3,374 243 67

31 4,449 4,077 3,977 4,168 249 83

16 4,776 4,785 4,782 4,781 5 95

8 4,052 3,834 4,165 4,017 168 80

4 4,540 4,504 4,577 4,540 37 90 These data demonstrate that (-)-epigallocatechin gallate and (-)-epicatechin gallate, both components of tea were effective at inhibiting bacterial attachment.

Another flavanol, Kaempferol was also tested in the Bacteria Adhesion Assay. Initial studies with this compound indicated that kaempferol, because of its color, interfered with the interpretation of the data generated using radio-isotope labelled microorganisms. The effect, called quenching, made it difficult to determine the number of organisms attached to the surface after treatment. An alternative method which utilized crystal violet for visualizing the attached bacteria, was used to stain the cells remaining on the microplate surface after treatment. The assay was conducted as previously described using bacteria which had not undergone radio-labeling. After removing all unattached bacteria by washing, all microtiter wells were filled with 125 μl of crystal violet solution (4 g/1). The dye remained in the wells for 20 minutes at room temperature. The excess dye was then removed by immersing the plates in a one liter beaker containing tap water. The water in the beaker was replaced during the rinsing process with fresh tap water. Immersion was continued until the tap water showed no residual purple color. Excess water was removed from the microtiter plates by inversion onto paper towels. After blotting, the plates were allowed to air dry for, at minimum, one hour. The cell-associated crystal violet was re-suspended in 150 μl of Sorenson's Buffer (50 mM Sodium Citrate pH 4.2, 50% Ethanol) and mixed for two hours. The amount of dye retained by the attached cells was determined using a Molecular Devices Thermomax microplate reader at 540 nm. The amount of dye retained is a function of the number of cells bound and it was compared to the amount of dye retained by untreated control cells. This comparison yielded percent attachment data very similar to that generated using radio-isotope labeled bacteria for the other compounds tested. The data using crystal violet is shown in Table VI.

Table VI

Crystal Violet Modified Bacterial Adhesion Assay Test Organism: Pseudomonas aeruginosa Avg. Standard %

Concentration (ppm) QD₅₄₀ QD₅₄₀ Deviation Attachment

Control:

1.567 1.729 1.661 1.565 1.445 1.696

1.641 1.804 1.521 1.625 0.112 Table VI. continued

Crystal Violet Modified Bacterial Adhesion Assay Test Organism: Pseudomonas aeruginosa

Avg. Standard %

Concentration (pprr QD₅₄₀ ΩD₅₄₀ Deviation Attachment

Kaempferol

500 0.875 0.763 0.961 0.866 0.099 53%

250 1.437 1.228 1.549 1.404 0.162 86%

125 2.037 1.885 1.845 1.922 0.101 118%

63 1.765 1.833 1.953 1.850 0.095 114%

31 1.479 1.603 1.711 1.598 0.116 98%

16 1.678 1.627 1.688 1.664 0.033 102%

These results demonstrate that Kaempferol is not as effective at inhibiting bacterial attachment compared to epigallocatechm gallate or epicatechin gallate.

To confirm that epigallocatechm gallate and epicatechin gallate would give comparable results to those obtained with the radio-labeled cells, the crystal violet modified Bacterial Adhesion Assay was conducted with these two compounds. The results appear in Table NIL

Table VII

Adhesion Assay to Test Efficacy of (-)-Epigallocatechin gallate and (-)-Epicatechin gallate Crystal Violet Modified Bacterial Adhesion Assay

Test Organism: Pseudomonas aeruginosa

Avg. Standard %

Concentration <ppm) QD₅₄₀ QD₅₄₀ Deviation Attachment Control:

1.237 1.447 1.210 1.335 1.556 1.570

1.613 1.505 1.539 1.446 0.150

-^•chin gallate

500 0.075 0.109 0.170 0.118 0.048 8

250 0.089 0.118 0.086 0.098 0.018 7

125 0.086 0.084 0.091 0.087 0.004 6

63 0.146 0.152 0.156 0.151 0.005 10

31 0.181 0.169 0.148 0.166 0.017 11

16 0.423 0.386 0.646 0.485 0.141 34 l gallate

500 0.073 0.170 0.065 0.082 0.022 6

250 0.087 0.139 0.088 0.105 0.030 7

125 0.094 0.113 0.091 0.099 0.012 7

63 0.148 0.186 0.137 0.157 0.026 11

31 0.429 0.416 0.346 0.397 0.045 27

16 0.739 0.786 0.651 0.725 0.069 50 These data show similar trends comparable to those data presented in Table V. As stated previously, kaempferol was not as effective as epigallocatechin gallate or epicatechin gallate at inhibiting the attachment of microbes to surfaces.

Further experiments were performed using (-)-catechin, (-)-epicatechin, and (-)- epigallocatechin in the Bacterial Adhesion Assay.

These results are reported in Table VIII. Radio-labeled cells were used for this assay.

Table VIII

Adhesion Assay Test Organism: Pseudomonas aeruginosa

Avg. Standard %

Concentration (ppm CPM CPM Deviation Attachment

Control:

4,671 5,304 4,831

5,030 4,874 5,179

5,035 5,323 5,089 5,037 218

(-)-Catechm

500 4,331 4,796 4,412 4,513 248 90

250 4,364 4,904 4,488 4,585 283 91

125 4,944 5,507 5,407 5,286 300 105

63 5,121 5,353 6,282 5,585 614 111

31 4,920 5,052 4,986 4,986 66 99

16 4,843 5,307 4,972 5,041 240 100

8 4,345 4,819 4,895 4,686 298 93

4 4,807 5,317 5,156 5,093 261 101

(-)-Epιcatechm

500 4,546 4,482 4,706 4,578 115 91

250 5,022 5,520 4,902 5,148 328 102

125 5,602 6,074 5,152 5,609 461 111

63 5,238 6,040 5,301 5,526 446 110

31 5,787 6,088 5,704 5,860 202 116

16 5,361 5,686 5,834 5,627 242 112

8 5,228 5,543 5,433 5,401 160 107

4 5,441 5,621 5,475 5,512 96 109

(-)-Epιgallocatechιn

500 3,352 3,406 3,281 3,346 63 66

250 3,883 3,886 3,668 3,812 125 76

125 4,505 4,273 4,388 4,389 116 87

63 4,524 4,611 4,571 4,569 44 91

31 4,141 4,536 4,067 4,248 252 84

16 5,109 4,600 4,522 4,744 319 94

8 4,678 4,145 4,170 4,331 301 86

4 4,849 4,730 4,564 4,714 143 94 None of these compounds except (-)-Epigallocatechin appears to be as effective at the concentrations tested.

Gallic acid was also tested in an attempt to determine if this functional group contributed to anti-sessile activity. Results are presented in Table IX.

Table IX

Adhesion Assay Test Organism: Pseudomonas aeruginosa

Avg. Standard %

Concentration (ppml CPM CPM Deviation Attachment

Control:

4,434 4,432 4,393 4,399 3,666 4,093

3,890 4,244 4,428 4,220 280

Gallic Acid

500 4,379 4,359 4,519 4,419 87 105

250 4,658 4,708 4,693 4,686 26 111

125 4,252 4,565 4,900 4,572 324 108

63 4,408 5,028 5,063 4,833 368 115

31 4,983 5,001 4,781 4,922 122 117

16 4,696 5,037 5,169 4,967 244 118

8 4,683 5,087 5,116 4,962 242 118

4 4,870 5,017 5,029 4,972 89 118

As indicated in these results, gallic acid did not show any anti-sessile activity.

While this invention has been described with respect to particular embodiments thereof, it is apparent that numerous other forms and modifications of this invention will be obvious to those skilled in the art. The appended claims and this invention generally should be construed to cover all such obvious forms and modifications which are within the true spirit and scope of the present invention.

Claims

Having thus described the invention, what we claim is:

1. A method for inhibiting the attachment of microbes to the surfaces of an aqueous system comprising adding to said aqueous system an effective inhibiting amount of flavanol compound to inhibit the attachment of the microbes to the surfaces of the aqueous system.

2. The method as claimed in claim 1 wherein said flavanol compound is derived from or contained in a tea fraction.

3. The method as claimed in claim 1 wherein said flavanol compound has the formula:

wherein R, is selected from H and OH, R₂ is selected from H, OH, and 0(3,4,5- trihydroxybenzoyl), and R₃ is selected from H or OH.

4. The method as claimed in claim 1 wherein said flavanol compound is selected from (-)-epicatechin gallate, (-)-epigallocatechin, (-)-epigallocatechin gallate, or kaempferol.

5. The method as claimed in claim 3 wherein said flavanol compound comprises mixtures of flavanol compounds.

6. The method as claimed in claim 3 wherein said flavanol compound comprises mixtures of flavanol compounds and tea fractions.

7. The method as claimed in claim 1 wherein said flavanol compound is (-)-epicatechin gallate.

8. The method as claimed in claim 1 wherein said flavanol compound is (-)-epigallocatechin.

9. The method as claimed in any one of claims 1-8 wherein said microbes are bacteria.

10. The method as claimed in any one of claims 1-8 wherein said microbe is Pseudomonas aeruginosa.

11. The method as claimed in any one of claims 1 -8 wherein said microbe is

Klebsiella pneumoniae.

12. The method as claimed in any one of claims 1-8 wherein said flavanol compound is added to said aqueous system in an amount ranging from about 5 parts to about 500 parts per million parts of said aqueous system.

13. The method as claimed in claim 2 wherein said tea fraction is added to said aqueous system in an amount ranging from about 5 parts to about 500 parts per million parts of said aqueous system.

14. The method as claimed in any one of claims 1-8 wherein said aqueous system is a papermaking system.

15. The method as claimed in any one of claims 1-8 wherein said aqueous system is a cooling water system.

16. The method as claimed in any one of claims 1-8 wherein said flavanol compound is applied directly to said surface.

17. The method as claimed in any one of claims 1-8 further comprising adding a biocide to said aqueous system.

18. The method as claimed in any one of claims 1-8 further comprising adding a surfactant to said aqueous system.