Use of lactams

Abstract

The present invention relates to the use of a lactam composition to improve toilet hygiene, which use comprises treating a toilet surface, wherein the composition comprises 0.0001 to 5 wt% of a lactam; which use relates to reducing malodour; or wherein the use relates to reducing undesirable urine staining in a toilet; wherein the lactam is of formula (I) or (II), wherein R1 and R2 are each independently selected from hydrogen, halogen, alkyl, cycloalkyl, alkoxy, oxoalkyl, alkenyl, heterocyclyl, heteroaryl, aryl and aralkyl; and R3 is selected from hydrogen, hydroxyl, alkyl, cycloalkyl, alkoxy, oxoalkyl, alkenyl, heterocyclyl, heteroaryl, cycloalkyl, aryl, aralkyl, -C(O)CR6=CH2 and (CH2) n N + (R a )3, wherein n is an integer from 1 to 16, preferably 2 to 8, and wherein each R a is independently H or C 1‑4 alkyl; R4 and R5 are independently selected from hydrogen, aryl, heterocyclyl, heteroaryl and arylalkyl; and R6 is selected from hydrogen and methyl; and R7 is selected from hydrogen and -C(O)CR6=CH2; and preferably at least one of R4 and R5 is hydrogen. (I) or (II).

Description

TECHNICAL FIELD

[0001] The present invention relates to improvements in the field of toilet hygiene, in particular to the use of lactam to improve toilet hygiene. BACKGROUND

[0002] Toilet hygiene is important to consumers. When human waste is disposed of in a toilet, consumers still want to maintain a hygienic toilet environment.

[0003] One way in which consumers recognise poor toilet hygiene is that the toilet emits an undesirable odour, classified as an undesirable malodour.

[0004] There is a need to improve toilet hygiene, for example by reducing malodour from a toilet, or by reducing undesirable crystal formation in a toilet. SUMMARY

[0005] We have found that toilet hygiene is improved by treating a toilet surface with a lactam composition.

[0006] In a first aspect, the present invention relates to the use of a lactam composition to improve toilet hygiene, the use comprising treating a toilet surface, wherein the composition comprises 0.0001-5 wt% of a lactam;

[0007] wherein the use relates to reducing malodour; or wherein the use relates to reducing undesirable uric scale formation in a toilet;

[0008] wherein the lactam has the formula (I) or (II):

[0009]

[0010] wherein:

[0011] R1and R2are each independently selected from hydrogen, halogen, alkyl, cycloalkyl, alkoxy, oxoalkyl, alkenyl, heterocyclyl, heteroaryl, aryl and aralkyl; and

[0012] R3is selected from hydrogen, hydroxyl, alkyl, cycloalkyl, alkoxy, oxoalkyl, alkenyl, heterocyclyl, heteroaryl, cycloalkyl, aryl, aralkyl, -C(O)CR6=CH2and (CH2) n N + (R a )3, wherein n is an integer from 1 to 16, preferably 2 to 8, and wherein each R a is independently H or C 1-4 alkyl;

[0013] R4and R5are independently selected from hydrogen, aryl, heterocyclyl, heteroaryl and arylalkyl; and

[0014] R6is selected from hydrogen and methyl; and

[0015] R7is selected from hydrogen and -C(O)CR6=CH2; and

[0016] Preferably, at least one of R4and R5is hydrogen.

[0017] Preferably, the lactam is present at a level of 0.0001 to 2.5 wt%, preferably 0.0001 to 1 wt%, more preferably 0.001 to 1 wt%.

[0018] The use includes the reduction of malodour. In particular, it is preferred that the reduction of malodour relates to the reduction of urine associated malodour, in particular the reduction of malodour associated with phenol and indole; or the use relates to the reduction of undesirable urine scale formation in a toilet. In particular, it is preferred that the reduction of undesirable urine scale formation in a toilet relates to the inhibition of P. Mirabilis.

[0019] The lactam is of formula (I) or (II):

[0020]

[0021] wherein:

[0022] R1and R2are each independently selected from hydrogen, halogen, alkyl, cycloalkyl, alkoxy, oxoalkyl, alkenyl, heterocyclyl, heteroaryl, aryl and aralkyl; and

[0023] R3is selected from hydrogen, hydroxyl, alkyl, cycloalkyl, alkoxy, oxoalkyl, alkenyl, heterocyclyl, heteroaryl, cycloalkyl, aryl, aralkyl, -C(O)CR6=CH2and (CH2) n N + (R a )3, wherein n is an integer from 1 to 16, preferably 2 to 8, and wherein each R a is independently H or C 1-4 alkyl;

[0024] R4and R5are independently selected from hydrogen, aryl, heterocyclyl, heteroaryl and arylalkyl; and

[0025] R6is selected from hydrogen and methyl; and

[0026] R7is selected from hydrogen and -C(O)CR6=CH2; and

[0027] Preferably at least one of R4and R5is hydrogen.

[0028] Preferably the lactam of formula (I) or (II), R1, R4and R5are H; R3is H or (CH2) n N +(CH3)3, wherein n is an integer from 1 to 16, preferably from 2 to 8; and R2is phenyl or mono-substituted phenyl; preferably R2is selected from the group consisting of phenyl, 4-fluorophenyl, 2-fluorophenyl, 4-chlorophenyl, 3-chlorophenyl, 4-bromophenyl and 4-methylphenyl.

[0029] Preferably the lactam is a lactam selected from the group consisting of:

[0030]

[0031] Preferably the lactam is selected from the group consisting of:

[0032]

[0033] Most preferably the lactam is

[0034] 4-(4-chlorophenyl)-5-methylene-pyrrol-2-one.

[0035] Preferably the lactam is delivered from an aqueous-based composition or an organic solvent-based composition.

[0036] Preferably the toilet surface to be treated is selected from the group consisting of ceramic, for example porcelain. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 Refers to Example 2 and shows the effect of lactam on malodor control (phenol).

[0038] Figure 2 Refers to Example 2 and shows the effect of lactam on malodor control (indole).

[0039] Figure 3 Refers to Example 3 and depicts images showing the effect of lactam on reducing Proteus mirabilis.

[0040] Figure 4 Refers to Example 3 and depicts graphs showing the effect of lactam on reducing Proteus mirabilis. DETAILED DESCRIPTION

[0041] As used herein, the indefinite articles "a" or "an" and their corresponding plural forms "at least one" and "one or more" mean "one or more than one" unless otherwise indicated.

[0042] It should be understood that all preferences are combinable unless otherwise stated.

[0043] Use for improving toilet hygiene

[0044] The use relates to reducing malodor, and / or the use relates to reducing undesirable urine stain formation in a toilet.

[0045] In particular, it is preferred that the reduction of malodour relates to a reduction of urine-related malodour, in particular to a reduction of malodour related to phenol and indole.

[0046] In case the use relates to a reduction of undesirable urine scale formation in a toilet, it is preferred that the reduction of undesirable urine scale formation in a toilet relates to the inhibition of Proteus mirabilis.

[0047] The formed urine scale is at least partially due to the formation of crystals. Proteus mirabilis produces urease that hydrolyzes urea to release carbon dioxide and ammonia, thereby increasing the pH of the urine and leading to the deposition of struvite and apatite crystal deposits. Inhibition of Proteus mirabilis is accompanied by inhibition of crystal formation.

[0048] Lactam

[0049] Lactams are cyclic amides. Preferred lactams are gamma-lactams having 5 ring atoms.

[0050] The lactam has the formula (I) or (II):

[0051]

[0052] wherein:

[0053] R1and R2are each independently selected from the group consisting of hydrogen, halogen, alkyl, cycloalkyl, alkoxy, oxoalkyl, alkenyl, heterocyclyl, heteroaryl, aryl, and aralkyl; and

[0054] R3is selected from the group consisting of hydrogen, hydroxyl, alkyl, cycloalkyl, alkoxy, oxoalkyl, alkenyl, heterocyclyl, heteroaryl, cycloalkyl, aryl, aralkyl, -C(O)CR6=CH2, and (CH2) n N + (R a )3, wherein n is an integer from 1 to 16, preferably from 2 to 8, and wherein each R a is independently H or C 1-4 alkyl;

[0055] R4and R5are independently selected from the group consisting of hydrogen, aryl, heterocyclyl, heteroaryl, and arylalkyl; and

[0056] R6is selected from the group consisting of hydrogen and methyl; and

[0057] R7is selected from the group consisting of hydrogen and -C(O)CR6=CH2; and

[0058] Preferably, at least one of R4and R5is hydrogen.

[0059] It is to be understood that where appropriate, groups can be optionally substituted. Optional substituents can include halogen, C 1-4 alkyl, C1-4 haloalkyl (e.g. CF3) and C 1-4 alkoxy.

[0060] alkyl can for example be C 1-12 alkyl, for example C 1-6 alkyl. Aryl can for example be C 6-10 aryl, for example phenyl.

[0061] Preferably, at least one of R1and R2is selected from heterocyclyl, heteroaryl, aryl and arylalkyl.

[0062] Preferably, R1is hydrogen. Preferably, R3is hydrogen or (CH2) n N + (R a )3, wherein n is an integer from 1 to 16, preferably 2 to 8, and wherein each R a is independently H or C 1-4 alkyl, more preferably R a is CH3; Preferably, R4is hydrogen. Preferably, R5is hydrogen. Preferably, R6is hydrogen. Preferably, R7is hydrogen. Preferably, R2is aryl or aralkyl. More preferably, R2is phenyl or substituted phenyl, for example mono-substituted phenyl. The substitution can be at the ortho, meta or para position. Preferred substituents include halogen and methyl. For example, but not limited to, R2may be selected from phenyl, 4-fluorophenyl, 2-fluorophenyl, 4-chlorophenyl, 3-chlorophenyl, 4-bromophenyl and 4-methylphenyl.

[0063] More preferably in the lactam of formula (I) or (II), R1, R4and R5are H; R3is H or (CH2) n N + (CH3)3, wherein n is an integer from 1 to 16, preferably 2 to 8; and R2is phenyl or mono-substituted phenyl; preferably R2is selected from phenyl, 4-fluorophenyl, 2-fluorophenyl, 4-chlorophenyl, 3-chlorophenyl, 4-bromophenyl and 4-methylphenyl.

[0064] Even more preferably the lactam has formula (I), R1, R4and R5are H; R3is H or (CH2) n N + (CH3)3, wherein n is an integer from 1 to 16, preferably 2 to 8; and R2is phenyl or mono-substituted phenyl; preferably R2is selected from phenyl, 4-fluorophenyl, 2-fluorophenyl, 4-chlorophenyl, 3-chlorophenyl, 4-bromophenyl and 4-methylphenyl.

[0065] In the case of cationic in nature, it can be used as is or suitably with a counterion (e.g. iodide).

[0066] Preferably the lactam is a lactam selected from the group consisting of:

[0067]

[0068] More preferably the lactam is selected from the group consisting of:

[0069]

[0070] Most preferably the lactam is:

[0071] 4-(4-chlorophenyl)-5-methylene-pyrrol-2-one.

[0072] In the case of cationic nature of the lactam, it can be used as such or suitably together with a counterion, such as iodide.

[0073] Level of lactam

[0074] Preferably the lactam is present at a level of 0.0001 to 2.5 wt.%, preferably 0.0001 to 1 wt.%. For example, the lactam can suitably be present at a level of 0.001 to 1 wt.% or even 0.01 to 1 wt.% or even 0.01 to 0.5 wt.%.

[0075] Composition

[0076] Preferably, the lactam is delivered from a water-based composition or an organic solvent-based composition, most preferably a water-based composition.

[0077] The composition comprises 0.0002 - 0.1 wt.%, preferably 0.001 - 0.1 wt.% of the lactam.

[0078] Toilet surface

[0079] Preferably, the toilet surface to be treated is selected from the group consisting of ceramic (e.g. porcelain).

[0080] Further ingredients

[0081] The composition can comprise further ingredients, such as surfactants, chelating agents, thickening agents, pH adjusting agents.

[0082] The application will be further described by the following non-limiting examples.

[0083] Examples

[0084] Example 1 - Preparation of examples of preferred lactams

[0085] Preparation of 4-(4-chlorophenyl)-5-hydroxy-5-methylfuran-2(5H)-one

[0086]

[0087] Combine 1-(4-chlorophenyl)propan-2-one (40.00 g, 34.75 mL, 237.2 mmol), glyoxylic acid monohydrate (32.75 g, 355.8 mmol), and phosphoric acid (69.74 g, 711.7 mmol) at room temperature, then heat to 85 °C overnight. After cooling to room temperature, pour the mixture into a mixture of water (500 mL) and ethyl acetate (500 mL). Separate the layers and extract the aqueous phase with ethyl acetate (500 mL). Wash the combined organic layers with a 1:1 mixture of water and brine (2 x 500 mL), dry (MgS04), and concentrate under reduced pressure to give 4-(4-chlorophenyl)-5-hydroxy-5-methylfuran-2(5H)-one (66.00 g, >100% yield) as a brown oil. This material is used without further purification in the next step.

[0088] Preparation of 4-(4-chlorophenyl)-5-hydroxy-5-methyl-1 H-pyrrol-2(5H)-one

[0089]

[0090] Dissolve 4-(4-chlorophenyl)-5-hydroxy-5-methylfuran-2(5H)-one (66.00 g, 293.8 mmol) in thionyl chloride (196.8 g, 120.0 mL, 1654 mmol) and heat at 40 °C for 1 h, then at 80 °C for 2 h. Concentrate the mixture under reduced pressure and azeotrope with 2-methyltetrahydrofuran (200 mL). Dilute the residue with 2-methyltetrahydrofuran (160 mL) and add the solution to a cooled, stirred mixture of 28% aqueous ammonia (180 mL) in 2-methyltetrahydrofuran (20 mL) at 0 °C. Warm the mixture to room temperature and stir overnight. Add water (100 mL) and ethyl acetate (200 mL) and separate the layers. Extract the aqueous phase with ethyl acetate (200 mL), dry (MgS04) the combined organic extracts, and concentrate under reduced pressure. Purify by dry flash column chromatography (5-60% ethyl acetate in heptane) to give 4-(4-chlorophenyl)-5-hydroxy-5-methyl-1H-pyrrol-2(5H)-one (23.18 g, 35% yield) as a cream colored solid.

[0091] 1 H NMR (400 MHz, d6-DMSO) 8.55 (br s, 1H), 7.88-7.83 (m, 2H), 7.51-7.46 (m, 2H), 6.37 (d, 1H), 6.32 (s, 1H), 1.45 (s, 3H)

[0092] UPLC (basic) 1.51 / 5.00 min, 100% purity, M+H + 224

[0093] MP 177 °C

[0094] Preparation of 4-(4-chlorophenyl)-5-methylene-1 H-pyrrol-2(5H)-one

[0095]

[0096] To a cooled solution of 4-(4-chlorophenyl)-5-hydroxy-5-methyl-1 H-pyrrol-2(5H)-one (10.00 g, 44.51 mmol) in dry dichloromethane (100 mL) at 0 °C was added a solution of boron trifluoride etherate (8.213 g, 7.142 mL, 57.87 mmol) in dry dichloromethane (45 mL) over 15 minutes. The mixture was stirred at 0 °C, then slowly warmed to room temperature and stirred for 2 hours. The reaction was quenched with ice water (100 mL) and the layers were separated. The aqueous layer was extracted with dichloromethane (100 mL) and the combined organic layers were washed with a 1 :1 mixture of water and saturated aqueous sodium bicarbonate solution (100 mL), dried (MgS04) and filtered. Silica was added to the filtrate and the mixture was stirred for 10 minutes, then filtered through a plug of silica, washing well with dichloromethane, then a 3:1 mixture of dichloromethane:diethyl ether. The fractions containing the desired product were combined and concentrated under reduced pressure. A precipitate formed upon concentration which was collected by filtration, washing with diethyl ether to give 4-(4-chlorophenyl)-5-methylene-1 H-pyrrol-2(5H)-one (5.25 g, 57% yield) as a cream coloured solid.

[0097] 1 H NMR (400 MHz, d6-DMSO) 10.10 (s, 1 H), 7.54-7.47 (m, 4H), 6.36 (s, 1 H), 5.04 (t, 1 H), 4.85 (s, 1 H)

[0098] UPLC (basic) 1.87 / 5.00 min, 100% purity, M+H + 206

[0099] MP 182 °C

[0100] Preparation of 5-hydroxy-5-methyl-4-(p-tolyl)furan-2(5H)-one

[0101]

[0102] (2S)-2-(4-(4,4-difluoro-3-oxo-3,4-dihydro-2H-inden-1-yl)phenyl)-5-oxopyrrolidine-1- carboxylic acid (Compound 1) was prepared according to the following scheme:

[0103] 1 H NMR (400 MHz, d6-DMSO) 7.86 (s, 1H), 7.75 (d, 2H), 7.28 (d, 2H), 6.59 (s, 1H), 2.32 (s, 3H), 1.61 (s, 3H)

[0104] Preparation of 5-hydroxy-5-methyl-4-(p-tolyl)-1 H-pyrrol-2(5H)-one

[0105]

[0106] (2S)-2-(4-(4,4-difluoro-3-oxo-3,4-dihydro-2H-inden-1-yl)phenyl)-5-oxopyrrolidine-1- carboxylic acid (Compound 1) was prepared according to the following scheme:

[0107] 1H NMR (400 MHz, d6-DMSO) 8.44 (br s, 1 H), 7.73 (d, 2H), 7.21 (d, 2H), 6.24 (s, 2H), 2.29 (s, 3H), 1.45 (s, 3H)

[0108] 13 C NMR (400 MHz, d6-DMSO) 170.4 (s, 1 C), 161.1 (s, 1 C), 139.8 (s, 1 C), 129.7 (s, 2C), 128.9 (s, 1 C), 128.2 (s, 2C), 1 19.1 (s, 1 C), 87.8 (s, 1 C), 26.7 (s, 1 C), 21.5 (s, 1 C)

[0109] UPLC (basic) 1.41 / 5.00 min, 100% purity, M+H + 204

[0110] MP 178 °C decomposition

[0111] Preparation of 5-methylene-4-(p-tolyl)-1 H-pyrrol-2(5H)-one

[0112]

[0113] To a cooled solution of 5-hydroxy-5-methyl-4-(p-tolyl)-lH-pyrrol-2(5H)-one (8.68 g, 42.7 mmol) in dry dichloromethane (87 mL) at 0 °C was added a solution of boron trifluoride etherate (6.85 g, 5.96 mL, 55.5 mol) in dry dichloromethane (40 mL) over 15 minutes. After 1 hour, the mixture was allowed to slowly warm to room temperature. After a further 3 hours, the reaction was diluted with dichloromethane (50 mL) and ice water (100 mL) and stirred for 10 minutes. The layers were separated, the organic layer washed with water (100 mL), a 1 : 1 mixture of water and saturated aqueous sodium bicarbonate (100 mL) and brine (100 mL), the organic layer filtered through celite, washing with dichloromethane. Any excess water was removed by pipette and the filtrate was then dried (MgS04) and concentrated under reduced pressure to a brown solid. The solid was stirred in hot dichloromethane (120 mL) for 15 minutes, then allowed to cool slowly to room temperature, then to 0 °C. The solid was collected by filtration to give 5-methylene-4-(p-tolyl)-lH-pyrrol-2(5H)-one (3.87 g, 49% yield) as a yellow solid. Silica was added to the filtrate and the mixture stirred for 10 minutes, then filtered through a plug of silica, washing well with dichloromethane, then with a 4: 1 mixture of dichloromethane: diethyl ether. The filtrate was concentrated under reduced pressure to give 5-methylene-4-(p-tolyl)-lH-pyrrol-2(5H)-one (0.58 g, 7%) as a yellow solid. Overall yield of 5-methylene-4-(p-tolyl)-lH-pyrrol-2(5H)-one (4.45 g, 56% yield).

[0114] 1 H NMR (400 MHz, d6-DMSO) 10.11 (br s, 1H), 7.35 (d, 2H), 7.25 (d, 2H), 6.25 (s, 1H), 5.01 (s, 1H), 4.85 (s, 1H), 2.31 (s, 3H)

[0115] UPLC (Basic) 1.83 / 5.00 min, 100% purity, M+H + 186

[0116] MP 200 °C decomposes

[0117] Example 2 - Odour control

[0118] An artificial urine medium was prepared using the composition described below and sterilised using a 0.22 pm filter.

[0119] The lactam used is denoted as "488" and is 4-(4-chlorophenyl)-5-methylene-pyrrol-2-one.

[0120]

[0121] The artificial urine media composition is as shown in Table 1.

[0122] Table 1

[0123]

[0124]

[0125] A consortium of bacterial cultures was prepared by mixing equal volumes of 10 8 A consortium of bacterial cultures was prepared by mixing equal volumes of 10

[0126] A consortium of bacterial cultures was prepared by mixing equal volumes of 10

[0127] GC-MS (SIM) conditions

[0128] Instrument: Perkin Elmer Clarus 500 Gas Chromatograph-Mass Spectrometer. GC conditions: GC column: CP WAX (30 m x 0.25 mm ID x 0.25 pm thickness); Carrier gas: Helium (1 mL / min); Oven program: Initial temperature 35 °C for 5 min; Ramp: 10 °C / min to 230 °C for 5 min; Run time: 29.5 min; Injection port temperature: 230 °C. MS conditions: Inlet line temperature: 200 °C; Electron energy: 70 eV; Source temperature: 180 °C; Multiplier voltage: 400 V

[0129] CombiPAL conditions

[0130] The sample vials were equilibrated at 60 degrees for 5 minutes. The sampling time was 2 minutes. The desorption time was 2 minutes. The post fiber conditioning time was 38 minutes. The total run time was 40 minutes.

[0131] Detection of off-odor molecules using GC / MS-SIM mode

[0132] Operating the GC / MS in SIM mode allows for increased sensitivity in detecting specific analytes. In this mode, the MS collects data for the masses of interest, rather than seeking all masses in a broad range. Because the instrument is set to seek only the masses of interest, it can be specific to the specific analytes of interest. Thus, they are analyzed using the SIM mode. Typically, more than two ions are monitored for each compound, and the ratio of these ions is unique to the analyte of interest. The ions monitored for each molecule are recorded in Table 2. To increase sensitivity, the mass scan rate and dwell time (the time spent observing at each mass) are adjusted. Using this method, we can detect phenol (retention time - 18.5) and indole (retention time - 22.4).

[0133] Table 2

[0134]

[0135] The odor produced from the sample in the absence of lactam was considered 100%. The amount of odor produced was recorded from the peak area of the MO peak. The odor produced in the presence of lactam was calculated in comparison to this peak. The peak areas of phenol and indole from the sample with just the bugs were recorded and considered 100%. The peak areas of these two peaks in the sample with lactam were recorded and the % reduction compared to the sample without lactam was compared and plotted in Figure 1 and 2 The data is also shown in Tables 3 and 4.

[0136] Table 3 - Odour control of lactam (phenol)

[0137] Composition % phenol Positive control 100 Positive control + lactam 400 ppm 28

[0138] Table 4 - Odour control of lactam (indole)

[0139] Composition % indole Positive control 100 Positive control + lactam 400 ppm 68

[0140] Example 3 - Proteus mirabilis inhibition and control of urea crystals

[0141] Multi-strain biofilm experiments were performed with P. mirabilis Hauser 1885 labelled with ds-red, P. aeruginosa PAOl -uw labelled with m-turquoise and S. aureus SH1000 labelled with eGFP. Each strain was adjusted to an OD600 of 0.05 in polystyrene 24 well plates (Greiner) in artificial urine medium, statically inoculated for 2 hours at 37°C and incubated for a further 22 hours at 70 rpm for a total of 72 hours. P. mirabilis was grown first, followed by P. aeruginosa and finally S. aureus for a total of 72 hours. The medium was carefully removed and a new bacterial suspension was gently added before adding a new inoculum. Cells were grown in artificial urine medium. Crystals were the product of urease activity of P. mirabilis and as the pH increased, the natural salt precipitated from the medium.

[0142] Artificial urine medium

[0143] The artificial urine medium was prepared according to Brooks and Keevil (1997). 1 g proteose peptone L37, 0.005 g yeast extract, 0.1 g lactic acid, 0.4 g citric acid, 2.1 g NaHC03, 10 g urea, 0.07 g uric acid, 0.8 g creatinine, 0.37 g CaCl2.2H20, 5.2 g sodium chloride, 0.0012 g FeS04.7H20, 3.2 g Na20^O.10H20, 0.95 g KH2P04, 1.2 g HK20^P, 1.3 g NH4CI were made up to 1 L with distilled water. The pH was adjusted to 6.5 using HC1 (molar concentration, e.g. 2.5M). Finally, the medium was filter sterilised through a 0.22 μιη membrane filter (Millipore).

[0144] Confocal laser scanning microscopy

[0145] Confocal laser scanning microscopy (CLSM) was used to visualise and generate Z-stack representations of mixed species biofilms. Display was performed using a Zeiss LSM 700 confocal laser scanning microscope (Carl Zeiss, Ltd, Welwyn, UK) controlled through the ZEN 2009 software platform (Carl Zeiss, Ltd, Welwyn, UK) using a 5x or l Ox objective.

[0146] Acquisition settings depend on fluorescence: pinhole 1.0, laser intensity 5-15%, gain between 450-700 for bio-fluorescent strains. However, once adjusted to optimize visualization, settings were never modified in the experiments in order to perform unbiased comparisons. Z-stack height was set for each acquisition in a new well, starting from the first point where no emission through the biofilm itself was detected (just before the beginning of the biofilm) until the point where emission was reached again, which was set as the last slice. Once set, the interval was kept constant in the experiments.

[0147] Biofilm quantification and analysis

[0148] Biofilm analysis using TECAN TM Infinity F200 PRO plate reader. In order to be able to analyze the Z-stack images generated with CLSM, 3D pictures were processed in Zen 2.3 SP1 (black) software (Carl Zeiss, Jena, Germany) using the maximum intensity projection function. This tool generates a two-dimensional (2D) image for each channel, composed of pixels containing the maximum value over all images of the Z-stack at a single pixel position. Each new picture was exported in tiff format (64 bits), without compression to Zen 2.3 (blue version). Analysis and quantification of the whole images were performed using the Measure function in ImageJ 1.52i software (Rueden et al., 2017) implemented with the Fiji plugin bundle (Schindelin et al., 2012). The data generated for each single image were the mean grey value, defined as the sum of the grey values of all pixels divided by the number of pixels, and the related standard deviation. The final data represent the mean value of the samples grown in a specific condition, as the mean of the mean grey values generated in the images collected for each well. In Figure 3 and 4 The resulting graphics are shown in Figures.

Claims

1. Use of a lactam composition to improve toilet hygiene, the use including treating toilet surfaces, wherein the composition comprises 0.0001 to 5% by weight of lactam; The aforementioned use relates to reducing the formation of unwanted urine stains in the toilet; The lactam described herein has formula (I) or (II): In the lactam of formula (I) or (II), R1, R4, and R5 are H; R3 is H or (CH2). n N + (CH3)3, where n is an integer from 1 to 16; R2 is a phenyl or a monosubstituted phenyl; and R7 is selected from hydrogen and -C(O)CR6=CH2, where R6 is selected from hydrogen and methyl.

2. The use according to claim 1, wherein the lactam is present at a level of 0.0001-2.5% by weight.

3. The use according to claim 1, wherein the lactam is present at a level of 0.0001-1% by weight.

4. The use according to claim 1, wherein the lactam is present at a level of 0.001-1% by weight.

5. The use according to any one of claims 1-4, wherein reducing unwanted urine stains in the toilet involves inhibiting Proteus mirabilis.

6. The use according to any one of claims 1-4, wherein n is an integer from 2 to 8.

7. The use according to any one of claims 1-4, wherein R2 is selected from phenyl, 4-fluorophenyl, 2-fluorophenyl, 4-chlorophenyl, 3-chlorophenyl, 4-bromophenyl and 4-methylphenyl.

8. The use according to any one of claims 1-4, wherein the lactam is selected from the group consisting of:

9. The use according to any one of claims 1-4, wherein the lactam is selected from: and / or 10. The use according to any one of claims 1-4, wherein the lactam is: 4-(4-Chlorophenyl)-5-methylene-pyrrole-2-one.

Images