WO2008041008A1

WO2008041008A1 - Test method for biocidal activity

Info

Publication number: WO2008041008A1
Application number: PCT/GB2007/003807
Authority: WO
Inventors: Ulrich W. Schwarz
Original assignee: Byotrol Plc
Priority date: 2006-10-06
Filing date: 2007-10-05
Publication date: 2008-04-10
Also published as: CN101553575A; GB0619778D0; EP2082058A1

Abstract

The invention relates to a method and kit of parts for testing the biocidal activity of a substance. The method comprises the steps of (i) preparing a composition comprising sugar and yeast, (ii) mixing at least a portion of the composition with the substance to be tested in a first container, (iii) incubating the container and its contents, and (iv) comparing the amount of fermentation in the container with a control to give an indication of the biocidal activity of the substance.

Description

TEST METHOD FOR BIOCIDAL ACTIVITY

The invention relates to a method for testing the biocidal activity of a substance, particularly a liquid substance using a yeast-based test.

Known methods for testing biocidal activity require complicated and time- consuming procedures often requiring elaborate experimental procedures and/or sophisticated and expensive equipment. Such complicated procedures are not suited for use in the field.

For example, WO 01/20020 describes a method for identifying compounds with herbicidal or growth regulator activity. The method involves addition of a compound to be tested to a culture of a yeast strain expressing one or more plant cycle control genes. This procedure is specific for assaying herbicide, pesticide, fungicide or plant growth regulator activity and requires the preparation of a specific yeast strain.

Knight et al describe a yeast-based cytotoxicity assay for environmental monitoring (J. Environ. Monit, 2004, 6, 71-79). The assay requires the use of genetically modified yeast cells and instruments for measuring fluorescence.

Neither of the above approaches is suited to a general biocide activity test which may be used in the field using readily available materials. The present invention addresses these requirements.

The listing or discussion of a prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

The enzymes in yeast are known to convert sugar (e.g. glucose, fructose) to ethanol and carbon dioxide by a process known as fermentation. Fermentation is used in baking, the release of carbon dioxide causing the dough to rise and the ethanol contributing to the bread's flavour. The present inventors have found that the fermentation process may be retarded by the addition of a biocide. Surprisingly, the volume of foaming caused by the fermentation may be reliably and accurately used as a means for testing the biocidal activity of a substance. This has led to the development of an easy to handle on site method for testing the biocidal activity of a substance.

The invention provides a method for testing the biocidal activity of a substance.

The method comprises the steps of: (i) preparing a composition comprising sugar and yeast;

(ii) mixing at least a portion of the composition with the substance to be tested in a first container;

(iii) incubating the container and its contents; and

(iv) comparing the amount of fermentation in the container with a control to give an indication of the biocidal activity of the substance.

Thus, the present invention may be used to screen the activity of biocides and/or compositions containing biocides. A "biocide" is a substance which is toxic to living organisms. A biocide which may typically be tested by the method of the present invention is an antimicrobial agent, by which we mean any chemical substance which can destroy and/or prevent reproduction of and or kill microorganisms. Examples of antimicrobial agents include bactericidal, fungicidal, algicidal, yeasticidal, moldicidal, germicidal, spermicidal, antibiotic, antiviral, herbicidal, insecticidal and rodenticidal agents, and mixtures thereof, particularly bactericidal, fungicidal, yeasticidal, moldicidal and germicidal agents and mixtures thereof. As used herein, the term "biocidal activity" should be construed accordingly.

The "substance" to be tested may be any solid or liquid. If the substance tested is a liquid, it may for example be a solution or a suspension of a potential or known biocide or biocidal composition. The use of a liquid substance or a solid substance which dissolves in the other components used in the test typically improves its mixing with the other components used in the test and thus provides more reliable results compared to using a solid substance. The liquid substance may be a solution of a potential biocide (e.g. an aqueous solution of a solid or biocidal liquid substance) or a composition containing one or more biocides.

The present invention may be used to test solid or liquid samples found in the field for biocidal substances and/or activity. For example, the "substance" to be tested may be sample of soil or water (e.g. river, pond, lake, stream or seawater) found in the field. Thus, the present invention is particularly useful for field work.

By the term "sugar", we include any monosaccharide, disaccharide or polysaccharide or mixtures thereof.

By the term "amount of fermentation", we include the amount of ethanol and/or carbon dioxide produced during the fermentation of the sugar by the yeast in the incubation step (iii).

Preferably, the composition of step (i) comprises a foaming agent, and in step (iv), the volume of foaming in the container is compared with a control to give an indication of the biocidal activity of the substance. By the term "foaming agent", we include any substance which has the ability to foam. Typically, such substances will be liquids having a surface tension sufficient to enable bubbles to form and will not be biocides and will not substantially affect the biocidal activity of a biocide. However, such substances may also be solids which dissolve in the other components of the composition to form a liquid having these properties. For example, the foaming agent may be a substance such as a surfactant (e.g. coconut diethanolamide) that has the ability to foam by itself and/or when mixed with water or another liquid (e.g. one or more of the other components that may be used according to the invention). Examples of substances which may be used as foaming agents include fatty acid esters such as mono & diglyceride of fatty acids, sodium dodecylsulfate, polyoxyethylene-sorbitan-monolaurate, polyalkylene glycolether and phosphoglyceride. The foaming agent may be an emulsion or a colloidal dispersion. For example, a preferred foaming agent is milk. Alternatively or additionally, the amount of carbon dioxide produced (by fermentation) in the container may be compared with a control to give an indication of the biocidal activity of the substance. The amount of carbon dioxide produced in the container is typically measured by a carbon dioxide detector, such as an infra-red carbon dioxide sensor or a carbon dioxide sensitive Drager tube. Other methods and detectors for measuring carbon dioxide known in the art may also be used.

It is also contemplated that the amount of ethanol produced (by fermentation) in the container may be compared with a control to give an indication of the biocidal activity of the substance. Thus, known methods and detectors for measuring ethanol may used in the present invention.

As noted above, the invention is suitable for fieldwork. Preferably, therefore, the present invention does not involve complicated procedures and/or equipment (e.g. heavy, bulky, fragile equipment or equipment which is sensitive to use in the field). In other words, the method and kit (see below) of the invention preferably are simple to perform and use. For example, comparison of the amount of fermentation is preferably carried out without potentially complicated detection methods such as comparing the pressure and/or volume of carbon dioxide evolved in the container and the control or the pH of the contents of the container and the control. It is also preferable to avoid the use of electronic equipment in the method of the invention. For example, if a foaming agent is used, the amount of fermentation (and thus ultimately the indication of biocidal activity) may be measured visually from the height of the foam.

The control referred to in step (iv) above may comprise repeating steps (i) and (iii) of the method of the invention, i.e. repeating the method of the invention but without the substance to be tested. In other words, the control preferably comprises adding a portion of the composition comprising sugar and yeast to a second container having substantially the same dimensions as the first container and incubating the second container and its contents. The amount of fermentation of the control may then be compared to the amount of fermentation in the first container containing the tested substance to give an indication of the biocidal activity of the substance.

Preferably, the control is carried out under substantially the same conditions as the test. In other words, preferably about the same amount of the same composition is used (e.g., using the same amount and type of yeast and sugar and optionally foaming agent) and the container is incubated for about the same amount of time at about the same temperature. If the test substance to be tested comprises a substantial volume of a liquid, approximately the same volume of a suitable solvent (e.g., water or foaming agent) may be added to the control to mimic the conditions of the test (e.g., so that the overall volume of liquid used in the control is about the same as the volume of liquid used in the test).

The method of the invention may be used to test and/or compare the biocidal activity of more than one substance or test for the presence of a biocidal substance in a material such as a soil or water sample. Alternatively, the method may be used to compare the biocidal activity of different concentrations of a substance or the presence and/or amount of a biocide in a material such as a soil or water sample. For example, the substance being tested may be diluted to a varying degree with water or another solvent. Preferably, of course, the method for testing the biocidal activity of a substance is carried out more than once, i.e. repeated one or more times on the same sample to confirm the results.

In other words, steps (i) to (iii) of the present invention may be repeated with one or more further substances and the amount of fermentation in each of the containers compared with a control.

Virtually any container may be used in the present invention. If the volume of foam is used to indicate the amount of fermentation, a container that is suitable for reliably comparing the volume of foam produced in the or each test and the control may be used in the present invention. For example, such containers used in the tests and the control may have substantially the same dimensions, preferably the same dimensions. By the phrase "substantially the same dimensions", we include the meaning that the dimensions of the containers are similar enough to allow reliable comparison of the volume of foaming between tests (e.g., simply by comparison of height of foaming), and hence the degree of biocidal activity.

The containers are preferably made of a rigid material such as plastic or glass. Preferably, the containers are open at one end to allow the components used in the test to be added easily to the containers. Optionally, such open containers may have a lid to allow the contents of the containers to be closed from the air. Preferably, however, the containers are open during the incubation step (iii).

The containers may be longer in one dimension than the other two dimensions. This is particularly useful when the volume of foam is used to indicate the amount of fermentation, as it facilitates comparison of the volume of foaming. Preferably, the containers have a height that is greater than their width or length. For example, the containers preferably have a diameter of from about 0.1 to about 10 cm and a height of from about 1 to about 50 cm. In such preferred containers, the height of the foaming in the containers gives an indication of the biocidal activity of the substance being tested in the container. Examples of suitable containers which may be used in the method of the invention include test tubes, beakers and conical flasks, preferably transparent tubes such as transparent test tubes.

Step (i) of the method of the invention concerns the preparation of the composition which is used to test the biocidal activity of the substance. The composition comprises yeast and sugar. The composition may also comprise water. Preferably, the composition comprises a foaming agent. In this case, the composition preferably is prepared by adding yeast to a solution of the sugar in the foaming agent. By doing so, osmotic damage to the yeast is thought to be avoided.

The composition may be prepared at ambient temperature or below. As used herein unless otherwise stated, by the term "ambient temperature", we include the meaning of any temperature found on-site in the field. For example, the composition may be prepared at a temperature of from about 0 to about 35 ⁰C, for example from about 1 to about 30⁰C, or from about 2 to about 25 ⁰C, such as from about 3 to about 20 ⁰C or from about 4 to about 15 ⁰C. In order to arrest or prevent fermentation of the composition before use, it may be stored below ambient temperatures (e.g. refrigerated) before use. Preferably, however, the composition is used immediately following its preparation. If required, the composition may be made into a suspension and if necessary further diluted by the addition of water.

In step (ii) of the method of the invention, a portion of the composition is mixed with the substance to be tested in a container as hereinbefore described. The substance to be tested may need to be diluted (e.g., with water) in the container prior to addition of the portion of the composition. Preferably, therefore, the portion of the composition is added to the container containing the substance to be tested. Preferably, the mixture of the composition and the substance to be tested is agitated before use, e.g. by sonication or shaking to properly mix the contents of the container. For example, for on-site field use, the mixture is preferably agitated by shaking.

The mixture is then incubated at a temperature suitable for fermentation of the sugar by the yeast. Typically, the mixture is incubated at a temperature of from 15 to 50 ⁰C. The temperature should be warm enough to allow fermentation to occur but not too warm so as to kill the yeast cells. Preferably, the mixture is incubated at a temperature of from about 20 to about 45 ⁰C, such as from about 23 to about 40⁰C, for example from about 25 to about 35 ⁰C (e.g. about 30 ⁰C).

The amount of time the mixture is incubated for is not critical and may range from about 1 to about 100 minutes or even longer. However, the incubation time should be the same as that used for the control and any other comparative test for the biocidal activity of a substance. Preferably, the mixture is incubated for about 5 to about 30 minutes, such from about 10 to about 20 minutes (e.g. about 15 minutes at, for example, about 30 °C. Typically, fermentation will occur for a defined time before stopping due to limited nutrients (e.g. sugar) and/or limited oxygen. For example, fermentation may be complete within about 15 minutes at about 30 ⁰C.

Following incubation, the amount of fermentation (for example, the volume of foaming) in the first container is compared to a control, and optionally any other test carried out. The amount of fermentation is representative of the biocidal activity of the substance.

The present invention also provides a kit of parts for carrying out the method of the invention. Thus, the invention provides a kit of parts comprising yeast and sugar. Preferably, the kit of parts also comprises a foaming agent and/or a carbon dioxide detector (such as those described hereinbefore). However, the kit preferably does not contain sensors for measuring the volume and/or pressure of carbon dioxide produced and/or sensors for measuring pH change associated with the evolution of carbon dioxide. Preferably, the kit does not contain electronic equipment.

The kit of the invention preferably also comprises two or more containers having substantially the same dimensions. The kit may also comprise a holder for one or more containers.

The kit may also comprise a measuring means (e.g. one or more rulers) to aid the comparison of the volume (e.g. height) of foaming in the preferred method of the invention wherein a foaming agent is used. Alternatively, the holder and/or the containers themselves may comprise such a measuring means (e.g. the containers and or holder may be graduated).

The kit may also comprise a fridge for cooling the other components of the kit and/or an incubator for carrying out step (iii) of the method of the invention.

Optionally, the kit may comprise a means for accurate addition of a liquid substance to be tested and/or a foaming agent and/or water if they are used in the method of the invention. Such a means may be a pipette of known volume, a syringe or any other suitable means capable of delivering a known and measurable volume of liquid.

Optionally, the yeast and sugar contained within the kit of parts may be premeasured into the containers.

The kit may also contain a biocide standard as positive control and sterile water as negative control. An example of a biocide standard is cocoalkyl dimethyl benzyl ammonium chloride, diluted to a known concentration with water as required.

The yeast which may be used in the method and kit of the invention is preferably baker's yeast, although other types of yeast, e.g. brewer's yeast, may be used. Preferably, fresh yeast is used, e.g. fresh baker's yeast. Dried or instant yeast may also be used. It is preferable to use a yeast that has not been genetically modified in any way.

Any type of milk may be used in the method and kit of the invention. However, it is preferable to use fresh milk, more preferably, fresh whole milk. Fresh whole milk with natural fat is preferred to low fat milk as low fat milk may lead to less foaming. It is preferable to use the same milk for the test(s) and for the control.

Any type of sugar may be used in the method and kit of the invention. Suitable sugars include monosaccharides such as glucose, fructose, galactose and ribose and disaccharides such as sucrose, maltose and lactose, preferably glucose. Preferably, the sugar used is powdered as this aids dissolution of the sugar in the milk. Any commercially available sugar may be used such as white granulated sugar, castor sugar or icing sugar.

The invention will now be described with reference to the following non-limiting Example.

Example In a conical flask, 3 g of powdered sugar was dissolved in 50 ml of cold milk (approximately 8 ⁰C). 15 g of fresh baker's yeast was added to the sugar solution and stirred to emulsify the yeast until a suspension of homogeneous colour was obtained. The suspension was stored at approximately 8 ⁰C for about 24 hours before use.

To 5 separate glass test tubes (height 10 cm, diameter 0.5 cm) was added a 0.5 ml sample of the biocide to be tested. The biocide tested was Byotrol A1616 F4L, the composition of which is set out below.

Al 616 PRODUCT (F4L Ready To Use)

CAS-No. COMPOUND Active Cone

6824-85-1 Benzyl-C_12-16-alkyldimethyl-, Chloride 0.07 %

7173-51-7 di-n-decyl dimethylammonium chloride 0.07 %

52-51-7 bronopol (INN) 0.05 %

27083-27-8 Polymeric Biguanide Hydrochloride 0.03 %

64-17-5 ethanol 0.13 %

990001-58-01 polydol 0.015 % water 99.64 %

The concentration of the five biocide samples varied from neat to a 1:79 dilution in distilled water. 1 ml of the yeast/sugar suspension was added to each of the five test tubes. To a further two glass test tubes (the control) was added 0.5 ml of the yeast/sugar suspension and 0.5 ml of a diluted yeast suspension (1 :2 in water).

The seven tubes were shaken gently and incubated at 37 ⁰C for approximately 15 minutes. The height to which the foam had risen in each tube was measured and the results are illustrated in Fig 1. The foam in the control had risen to approximately 8 cm (7cm for the tube in which the yeast concentration was reduced by dilution) and the height of the foam in the 5 samples tested increased approximately linearly from the neat biocide to the 1 :79 diluted biocide.

Claims

1. A method for testing the biocidal activity of a substance comprising the steps of: (i) preparing a composition comprising sugar and yeast;

(iii) incubating the container and its contents; and

2. A method according to claim 1 wherein the composition of step (i) comprises a foaming agent, and in step (iv), the volume of foaming in the container is compared with a control to give an indication of the biocidal activity of the substance.

3. A method according to claim 2 wherein height of the foam in the containers gives an indication of the biocidal activity of the or each substance.

4. A method according to any of the preceding claims wherein step (iv), the amount of carbon dioxide produced in the container is compared with a control to give an indication of the biocidal activity of the substance.

5. A method according to claim 4 wherein the amount of carbon dioxide produced is measured by a carbon dioxide detector.

6. A method according to any of the preceding claims wherein the control comprises incubating at least a portion of the composition in a second container having substantially the same dimensions as the first container.

7. A method according to any of the preceding claims wherein the composition is prepared at a temperature of from about 0 to about 35 ⁰C.

8. A method according to any of the preceding claims wherein the incubation is carried out a temperature of from about 15 to about 45 ⁰C.

9. A method according to any of the preceding claims comprising mixing one or more further portions of the composition separately with one or more further substances to be tested in one or more respective further containers each having substantially the same dimensions as the or each container.

10. A method according to any of the preceding claims, wherein the substance is a liquid.

11. A method according to any of the preceding claims wherein the containers are rigid.

12. A method according to claim 11 wherein the containers have a diameter of from about 0.1 to about 10 cm and a height of from about 1 to about 50 cm.

13. A method according to any of the preceding claims wherein the containers are shaken before incubation.

14. A method according to any of the preceding claims wherein the composition is stored at a temperature of from about 0 to about 15 ⁰C before use.

15. A method according to any of the preceding claims wherein the yeast is fresh baker's yeast.

16. A method according to any of claims 2 to 15 wherein the foaming agent is milk.

17. A method according to claim 16 wherein the milk is fresh whole milk.

18. A method according to any of the preceding claims wherein the sugar is powdered.

19. A method according to any of claims 2 to 18 wherein the composition is prepared by adding the yeast to a solution of the sugar in the foaming agent.

20. A method according to any of the preceding claims wherein the first container also contains water for dilution.

21. A kit of parts for testing the biocidal activity a substance comprising yeast and sugar.

22. A kit of parts according to claim 21 comprising a foaming agent.

23. A kit of parts according to claim 22 wherein the foaming agent is milk.

24. A kit of parts according to any of claims 21 to 23 comprising a carbon dioxide detector.

25. A kit of parts according to any of claims 21 to 24 comprising two or more containers having substantially the same dimensions.

26. A kit of parts according to claim 25 comprising a holder for the containers.

27. A kit of parts according to any of claims 21 to 26 comprising a biocide standard and/or sterile water.

28. A kit of parts according to any of claims 21 to 27 comprising an incubator.

29. A kit of parts according to any of claims 21 to 28 comprising a fridge.

30. A kit of parts according to any of claims 21 to 29 comprising a means capable of delivering a known and measurable volume of liquid.

31. A kit of parts according to any of claims 21 to 30 comprising a means for measuring volume of foam.

32. A kit of parts according to any of claims 25 to 31 wherein the containers are tubes having substantially the same diameter.

33. A kit of parts according to claim 32 wherein the tubes have a diameter of from about 0.1 to about 10 cm and a height of from about 1 to about 50 cm.

34. A kit of parts according to any of claims 21 to 33 wherein the yeast is fresh baker's yeast.

35. A kit of parts according to any of claims 23 to 34 wherein the milk is fresh whole milk.

36. A kit of parts according to any of claims 21 to 35 wherein the sugar is powdered.

37. The use of a kit of parts according to any of claims 21 to 36 for testing the biocidal activity of a substance.

38. The use of a composition comprising yeast and sugar for testing the biocidal activity of a substance.

39. The use according to claim 38 wherein the composition comprises a foaming agent.

40. A method for testing the biocidal activity of a substance generally as herein described with reference to the Examples.

41. A kit of parts for testing the biocidal activity a substance generally as herein described.

42. Use of a kit of parts for testing the biocidal activity a substance generally as herein described.

43. Use of a kit of parts for testing the biocidal activity a substance generally as herein described with reference to the Examples.