EP4259765A1

EP4259765A1 - Use of lactam

Info

Publication number: EP4259765A1
Application number: EP21814687.6A
Authority: EP
Inventors: Panagiotis KOTSAKIS; Srilaxmi Venkata Medepalli; Neil James Parry
Original assignee: Unilever Global IP Ltd; Unilever IP Holdings BV
Current assignee: Unilever Global IP Ltd; Unilever IP Holdings BV
Priority date: 2020-12-09
Filing date: 2021-11-05
Publication date: 2023-10-18
Anticipated expiration: 2041-11-05
Also published as: CN116583586A; CN116583586B; WO2022122266A1; EP4259765B1

Abstract

The invention relates to the use a lactam composition to improve toilet hygiene, the use comprising treatment of a toilet surface, wherein the composition comprises from 0.0001 to 5 wt.% of a lactam; wherein the use involves reduction of malodour; or wherein the use involves reduction of unwanted urine scale formation in the 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 aralalkyl; and R₃ is selected from hydrogen, hydroxyl, alkyl, cycloalkyl, alkoxy, oxoalkyl, alkenyl, heterocyclyl, heteroaryl, cycloalkyl, aryl, aralalkyl, -C(O)CR₆=CH₂, and (CH₂)_nN⁺(R^a)₃, where n is an integer from 1 to 16, preferably 2 to 8, and where each R^a is independently H or C_1-4alkyl; R₄and 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.

Description

USE OF LACTAM

Field of Invention

The invention relates to an improvement in the field of toilet hygiene, in particular to the use of a lactam to improve toilet hygiene.

Background of the Invention

Toilet hygiene is important to consumers. While human waste is disposed of in the toilet, consumers still wish to maintain a hygienic toilet environment.

One way that consumers recognise poor toilet hygiene is that the toilet exudes an unwanted smell, classified as unwanted malodour.

There is a need to improve toilet hygiene, for example, by reducing malodour of the toilet, or by reducing unwanted crystal formation in the toilet.

Summary of the Invention

We have found that by using a lactam composition to treat a toilet surface, the toilet hygiene is improved.

The invention relates in a first aspect to the use of a lactam composition to improve toilet hygiene, the use comprising treatment of a toilet surface, wherein the composition comprises from 0.0001 to 5 wt.% of a lactam; wherein the use involves reduction of malodour; or wherein the use involves reduction of unwanted urine scale formation in the toilet; wherein the lactam is of formula (I) or (II): wherein:

Ri and R2 are each independently selected from hydrogen, halogen, alkyl, cycloalkyl, alkoxy, oxoalkyl, alkenyl, heterocyclyl, heteroaryl, aryl and aralalkyl; and

R3 is selected from hydrogen, hydroxyl, alkyl, cycloalkyl, alkoxy, oxoalkyl, alkenyl, heterocyclyl, heteroaryl, cycloalkyl, aryl, aralalkyl, -C(O)CR6=CH₂, and (CH₂)_nN⁺(R^a)₃, where n is an integer from 1 to 16, preferably 2 to 8, and where each R^a is independently H or C1-4 alkyl;

R4 and R₅ are independently selected from hydrogen, aryl, heterocyclyl, heteroaryl, and arylalkyl; and

Re is selected from hydrogen and methyl; and

R₇ is selected from hydrogen and -C(O)CRe=CH₂; and preferably, at least one of R4 and R₅ is hydrogen.

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

The use involves reduction of malodour. In particular, preferably where the reduction of malodour involves the reduction of malodour associated with urine, in particular relating to the reduction of malodour associated with phenol and indole; or the use involves reduction of unwanted urine scale formation in the toilet. In particular, preferably where the reduction of unwanted urine scale formation in the toilet involves the inhibition of P. mirabilis.

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 aralalkyl; and R3 is selected from hydrogen, hydroxyl, alkyl, cycloalkyl, alkoxy, oxoalkyl, alkenyl, heterocyclyl, heteroaryl, cycloalkyl, aryl, aralalkyl, -C(O)CR6=CH₂, and (CH₂)_nN⁺(R^a)₃, where n is an integer from 1 to 16, preferably 2 to 8, and where each R^a is independently H or C1-4 alkyl;

Re is selected from hydrogen and methyl; and

Preferably the lactam of formula (I) or (II), R1, R₄ and R₅ are H; R₃ is H, or (CH₂)_nN⁺(CH₃)₃, where n is an integer from 1 to 16, preferably 2 to 8; and R₂ is a phenyl group, or a monosubstituted phenyl group; preferably R₂ is selected from phenyl, 4-fluorophenyl, 2- fluorophenyl, 4-chlorophenyl, 3-chlorophenyl, 4-bromophenyl and 4-methylphenyL

Preferably the lactam is a lactam selected from:

Preferably the lactam is selected from:

Most preferably the lactam is

4-(4-chlorophenyl)-5-methylene-pyrrol-2-one

Preferably the lactam is delivered from an aqueous based composition, or an organic solvent based composition.

Preferably the toilet surface to be treated is selected from ceramics, for example porcelain.

Description of the Figures

Figure 1 relates to example 2 and shows the effect of malodour control (phenol) by lactam

Figure 2 relates to example 2 and shows the effect of malodour control (indole) by lactam

Figure 3 relates to example 3 and depicts images showing the effect of reduction of P.

Mirabilis by lactam

Figure 4 relates to example 3 and depicts a graph showing the effect of reduction of P. Mirabilis by lactam

Detailed Description of the Invention

The indefinite article "a" or "an" and its corresponding definite article "the" as used herein means at least one, or one or more, unless specified otherwise.

It will be appreciated that, except where expressly provided otherwise, all preferences are combinable. Uses for improvement of toilet hygiene

The use involves reduction of malodour, and/or the use involves reduction of unwanted urine scale formation in the toilet.

In particular, preferably where the reduction of malodour involves the reduction of malodour associated with urine, in particular relating to the reduction of malodour associated with phenol and indole.

Where the use involves reduction of unwanted urine scale formation in the toilet, preferably where the reduction of unwanted urine scale formation in the toilet involves the inhibition of P. mirabilis.

The urine scale that form is at least in part due to the formation of crystals. Proteus mirabilis produces ureases that hydrolyse urea releasing carbon dioxide and ammonia, increasing the pH of urine, and leading to struvite and apatite crystalline deposits. Proteus mirabilis inhibition is concomitant with crystal formation inhibition.

Lactam

A lactam is a cyclic amide. Preferred lactams are y-lactams which have 5 ring atoms.

The lactam is of formula (I) or (II): wherein:

Ri and R2 are each independently selected from hydrogen, halogen, alkyl, cycloalkyl, alkoxy, oxoalkyl, alkenyl, heterocyclyl, heteroaryl, aryl and aralalkyl; and R3 is selected from hydrogen, hydroxyl, alkyl, cycloalkyl, alkoxy, oxoalkyl, alkenyl, heterocyclyl, heteroaryl, cycloalkyl, aryl, aralalkyl, -C(O)CR6=CH₂, and (CH₂)_nN⁺(R^a)₃, where n is an integer from 1 to 16, preferably 2 to 8, and where each R^a is independently H or C1-4 alkyl;

Re is selected from hydrogen and methyl; and

It will be appreciated that, where appropriate groups may be optionally substituted. Optional substituents may include halogens, Ci-4alkyl, Ci-4haloalkyl (for example, CF₃) and Ci-4alkoxy.

Alkyls may, for example, be Ci -1₂alkyls, such as Ci-ealkyls. Aryls may, for example, be Ce- aryls, for example, phenyls.

Preferably, at least one of R1 and R₂ is selected from heterocyclyl, heteroaryl, aryl and arylalkyl.

Preferably, R1 is hydrogen. Preferably, R₃ is hydrogen, or (CH₂)_nN⁺(R^a)₃, where n is an integer from 1 to 16, preferably 2 to 8, and where each R^a is independently H or C1-4 alkyl, more preferably R^a is CH₃; Preferably, R4 is hydrogen. Preferably, R₅ is hydrogen. Preferably, Re is hydrogen. Preferably, R₇ is hydrogen. Preferably, R₂ is aryl or aralalkyl. More preferably, R₂ is a phenyl group or a substituted phenyl group, for example, a monosubstituted phenyl group. Substitution may be ortho, meta, or para. Preferred substituents include halogen and methyl. For example, and without limitation, R₂ may be selected from phenyl, 4-fluorophenyl, 2-fluorophenyl, 4-chlorophenyl, 3-chlorophenyl, 4-bromophenyl and 4-methylphenyl.

More preferably in the lactam of formula (I) or (II), R1, R₄ and R₅ are H; R₃ is H, or (CH₂)_nN⁺(CH₃)₃, where n is an integer from 1 to 16, preferably 2 to 8; and R₂ is a phenyl group, or a mono-substituted phenyl group; preferably R₂ is selected from phenyl, 4- fluorophenyl, 2-fluorophenyl, 4-chlorophenyl, 3-chlorophenyl, 4-bromophenyl and 4- methylphenyl. Even more preferably the lactam is of formula (I), Ri, FU and R₅ are H; R₃ is H, or (CH₂)_nN⁺(CH₃)₃, where n is an integer from 1 to 16, preferably 2 to 8; and R₂ is a phenyl group, or a mono-substituted phenyl group; preferably R₂ is selected from phenyl, 4- fluorophenyl, 2-fluorophenyl, 4-chlorophenyl, 3-chlorophenyl, 4-bromophenyl and 4- methylphenyl.

Where the lactam is cationic in nature, it can be used as such, or suitably with a counterion (e.g. iodide) Preferably the lactam is a lactam selected from:

More preferably the lactam is selected from:

Most preferably the lactam is chlorophenyl)-5-methylene-pyrrol-2-one.

Where the lactam is cationic in nature, the cation can be used or with a suitable counterion (e.g. iodide).

Levels of lactam

Preferably the lactam is present at a level of from 0.0001 to 2.5 wt.%, preferably from 0.0001 to 1 wt.%. For example, the lactam may be suitably present at levels of 0.001 to 1 wt.%, or even 0.01 to 1 wt.%, or even 0.01 to 0.5 wt.%.

Compositions

Preferably the lactam is delivered from an aqueous based composition, or an organic solvent based composition, most preferably an aqueous based composition.

The composition comprises from 0.0002 to 0.1 wt.%, preferably from 0.001 to 0.1 wt.% of a lactam.

Toilet Surface

Preferably the toilet surface to be treated is selected from ceramics (e.g. porcelain).

Further

The composition may comprise further ingredients such as surfactants, chelating agents, thickeners, pH modifiers.

The invention will be further described with the following non -limiting examples.

Example 1 - Preparation of examples of preferred lactams Preparation of 4-(4-chlorophenyl)-5-hvdroxy-5-methylfuran-2(5H)-one

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, 71 1 .7 mmol) were combined at room temperature before heating to 85 °C overnight. After cooling to room temperature, the mixture was poured into a mixture of water (500 mL) and ethyl acetate (500 mL). The layers were separated and the aqueous phase extracted with ethyl acetate (500 mL). The combined organic layers were washed with a 1 :1 mixture of water and brine (2 x 500 mL), dried (MgSC ) and concentrated under reduced pressure to yield 4-(4- chlorophenyl)-5-hydroxy-5-methylfuran-2(5H)-one (66.00 g, >100% yield) as a brown oil. The material was used in the next step without further purification.

Preparation of 4-(4-chlorophenyl)-5-hvdroxy-5-methyl-1 H-pyrrol-2(5H)-one

4-(4-Chlorophenyl)-5-hydroxy-5-methylfuran-2(5H)-one (66.00 g, 293.8 mmol) was dissolved in thionyl chloride (196.8 g, 120.0 mL, 1654 mmol) and heated at 40 °C for 1 hour, then 80 °C for 2 hours. The mixture was concentrated under reduced pressure and azeotroped with 2-methyltetrahydrofuran (200 mL). The residue was diluted with 2-methyltetrahydrofuran (160 mL) and this solution added to a cooled stirring mixture of 28% ammonia in water (180 mL) in 2-methyltetrahydrofuran (20 mL) at 0 °C. The mixture was warmed to room temperature and stirred overnight. Water (100 mL) and ethyl acetate (200 mL) were added and the layers separated. The aqueous phase was extracted with ethyl acetate (200 mL), and the combined organic extracts dried (MgSC>4) and concentrated under reduced pressure. Purification by dry flash column chromatography (5-60% ethyl acetate in heptane) yielded 4-(4-chlorophenyl)-5-hydroxy-5-methyl-1 H-pyrrol-2(5H)-one (23.18 g, 35% yield) as a cream coloured solid. ¹H NMR (400 MHz, d₆-DMSO) 8.55 (brs, 1 H), 7.88-7.83 (m, 2H), 7.51 -7.46 (m, 2H), 6.37 (d,

1 H), 6.32 (s, 1 H), 1.45 (s, 3H)

UPLC (Basic) 1.51/5.00 min, 100% purity, M+H+ 224

MP 177 ^eC

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

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 diethyl 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, before slowly warming to room temperature and stirring for 2 hours. The reaction was quenched with ice-water (100 mL) and the layers separated. The aqueous layer was extracted with dichloromethane (100 mL), and the combined organic layers washed with a 1 :1 mixture of water and saturated aqueous sodium hydrogen carbonate solution (100 mL), dried (MgSCU) and filtered. Silica was added to the filtrate and the mixture stirred for 10 minutes before filtering through a plug of silica, washing through with dichloromethane followed by a 3:1 mixture of dichloromethane:diethyl ether. Fractions containing the desired product were combined and concentrated under reduced pressure. Upon concentration a precipitate formed, which was collected by filtration, washing with diethyl ether, to yield 4-(4-chlorophenyl)-5-methylene-1 H-pyrrol-2(5H)-one (5.25 g, 57% yield) as a cream coloured solid.

¹H 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)

UPLC (Basic) 1 .87/5.00 min, 100% purity, M+H⁺ 206 MP 182 ^eC Preparation of 5-hvdroxy-5-methyl-4-(p-tolyl)furan-2(5H)-one

1 -(p-Tolyl)propan-2-one (25.00 g, 24.00 mL, 168.7 mmol), glyoxylic acid monohydrate (23.29 g, 253.0 mmol) and phosphoric acid (49.60 g, 506.1 mmol) were combined at room temperature before heating at 90 °C overnight. After cooling to room temperature, the mixture was poured into a stirring mixture of ice-water (400 mL) and ethyl acetate (400 mL). The layers were separated and the organic phase washed with water (100 mL), dried (MgSC ) and concentrated under reduced pressure. The mixture was azeotroped with 2- methyltetrahydrofuran (50 mL) to yield 5-hydroxy-5-methyl-4-(p-tolyl)furan-2(5H)-one (16.50 g, 48% yield) as a brown solid.

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

Preparation of 5-hvdroxy-5-methyl-4-(p-tolyl)-1 H-pyrrol-2(5H)-one 50 - 80 C

5-Hydroxy-5-methyl-4-(p-tolyl)furan-2(5H)-one (16.50 g, 80.80 mmol) was dissolved in thionyl chloride (48.06 g, 29.47 mL, 404.0 mmol) and heated at 50 °C for 1 hour, before heating at reflux for 1 hour. After cooling to room temperature, the mixture was concentrated under reduced pressure and azeotroped with 2-methyltetra-hydrofuran (2 x 50 mL). The residue was diluted with 2-methyltetrahydrofuran (60 mL) and this solution added to a cooled stirring mixture of 28% ammonia in water (55 mL, 808.0 mol) in 2-methyltetrahydrofuran (10 mL) at 0 °C. The mixture was warmed to room temperature and stirred overnight. 2- Methyltetrahydrofuran was removed under reduced pressure, and the residue diluted with water (200 mL) and diethyl ether (100 mL) and the mixture stirred for 20 minutes at room temperature. The solids were collected by filtration and stirred in water (100 mL) and diethyl ether (50 mL) at room temperature for 10 minutes. The solids were collected by filtration and washed with water, diethyl ether and dried under vacuum at 50 °C to yield 5-hydroxy-5- methyl-4-(p-tolyl)-1 H-pyrrol-2(5H)-one (10.49 g, 31 % yield) as a light beige solid.

¹H NMR (400 MHz, d₆-DMSO) 8.44 (brs, 1 H), 7.73 (d, 2H), 7.21 (d, 2H), 6.24 (s, 2H), 2.29

(s, 3H), 1.45 (s, 3H)

¹³C 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), 119.1 (s, 1 C), 87.8 (s, 1 C), 26.7 (s, 1 C), 21 .5 (s, 1 C)

UPLC (Basic) 1.41/5.00 min, 100% purity, M+H+ 204

MP 178 ^eC Decomposition

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

To a cooled solution of 5-hydroxy-5-methyl-4-(p-tolyl)-1 H-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 diethyl etherate (6.85 g, 5.96 mL, 55.5 mmol) 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 and the organic layer washed with water (100 mL), a 1 :1 mixture of water and saturated aqueous sodium hydrogen carbonate solution (100 mL) and brine (100 mL) and the organic layer filtered through Celite, washing with dichloromethane. Any excess water was removed by pipette before drying the filtrate (MgSC ) and concentrating under reduced pressure to a brown solid. The solids were stirred in hot dichloromethane (120 mL) for 15 minutes before slowly cooling to room temperature and then 0 °C. The solids were collected by filtration to yield 5-methylene-4-(p- tolyl)-1 H-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 before filtering through a plug of silica, washing through with dichloromethane and then a 4:1 mixture of dichloromethane:diethyl ether. The filtrate was concentrated under reduced pressure to yield 5-methylene-4-(p-tolyl)-1 H-pyrrol- 2(5H)-one (0.58 g, 7%) as a yellow solid. Total yield of 5-methylene-4-(p-tolyl)-1 H-pyrrol- 2(5H)-one (4.45 g, 56% yield).

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

UPLC (Basic) 1.83/5.00 min, 100% purity, M+H⁺ 186 MP 200 ^eC Decomposition

Example 2 - Malodour Control

Artificial urine medium was prepared using the below mentioned composition and filter sterilized using a 0.22 pm filter.

The lactam used was denoted as ‘488’ and is 4-(4-chlorophenyl)-5-methylene-pyrrol-2-one. The artificial urine medium composition was shown in table 1 .

Table 1

A consortium of bacterial cultures was prepared by mixing equal volumes of 10⁸ cells/ ml of

M. morganii, S. agalactiae, C. koseri, E. faecalis, P. mirabilis, P. vulgaris, E. coli.

A 1 : 40 dilution mix of bacterial culture mix and sterile artificial urine medium was prepared in bulk and 5 ml of this mix was transferred to sterile GC vials. 1 % stock of Lactam 488 was prepared in DMSO. Different concentrations of lactam 488 corresponding to 50, 200 and 400 ppm was added to the above prepared GC vials in duplicates. A positive control was set up simultaneously without addition of Lactam 488. The vials were incubated at 37°C for 48 hours. After 48 hours, the vials were subjected to GC analysis. Sample vials were placed in CombiPAL vial holder. Auto SPME accessory was used to perform the analysis.

GC-MS (SIM) conditions Instrument: Perkin Elmer Clarus 500 Gas chromato graph -Mass spectrometer. GC Conditions were: GC column: CP WAX (30m x 0.25 mm ID x 0.25 pm thickness); carrier gas: Helium (1 mL/min); oven program: Initial temperature 35°C hold for 5 min; ramp: 10°C per minute to 230°C hold for 5 min; run time: 29.5 min; Injection port temperature: 230°C. MS Conditions were: Inlet line Temperature: 200°C; Electron Energy: 70eV; Source Temperature: 180°C; Multiplier voltage: 400 V

CombiPAL conditions

The sample vial was equilibrated at 60 degrees for 5 minutes. Sampling time was 2 minutes. Desorption time is 2 minutes. Post fiber conditioning time is 38 minutes. Total run time is 40 minutes.

Detection of Malodour molecules using GC/MS-SIM Mode

Operation of a GC/MS in SIM mode allows for detection of specific analytes with increased sensitivity. In this mode the MS gathers data for masses of interest rather than looking for all masses over a wide range. Because the instrument is set to look for only masses of interest it can be specific for a particular analyte of interest. Hence, they were analysed using SIM mode. Typically, two more than two ions are monitored per compound and the ratios of those ions will be unique to the analyte of interest. The ions monitored for each molecule is captured in Table 2. In order to increase sensitivity, the mass scan rate and dwell times (the time spent looking at each mass) are adjusted. Using this method, we could detect Phenol (Retention time-18.5) and Indole (Retention time- 22.4).

Table 2

The odour generated from the sample without Lactam was considered as 100%. The amount of odour generated is noted down from the peak area of the MO peaks. In comparison to this peak, the odour generated in presence of Lactam was calculated. The peak areas of Phenol and Indole from the sample only with bugs were noted down and considered to be 100%. The peak areas of these two peaks in the sample with Lactam was noted down and % reduction with respect to the sample without lactam was compared and plotted in a graph shown in figures 1 and 2 with the data also shown in tables 3 and 4. Table 3 - Malodour control (phenol) by lactam

Table 4 - Malodour control (indole) by lactam

3 - Proteus mirabilis inhibition and urea control

A multi-strain biofilm experiment was carried out with ds-red labelled P. mirabilis Hauser 1885, m-turquoise labelled P. aeruginosa PAO1 -uw and eGFP labelled S. aureus SH1000. The OD600 for each strain was adjusted for the inoculum at 0.05 with artificial urine medium in polystyrene 24-well plates (Greiner), left seeding for 2 h statically at 37 °C and incubated for a further 22 h at 70 rpm for a total of 72 h. Pr. mirabilis was grown first, followed by P. aeruginosa and finally S. aureus for a total of 72 h. Before addition of the new inoculum, the medium was carefully removed, and the new bacterial suspension gently added. Cells were cultured in artificial Urine. The crystals are product of the urease activity of P. mirabilis and natural precipitation of salts from medium when pH increases.

Artificial Urine Medium

Artificial Urine Medium was prepared following Brooks and Keevil (1997). One gram of peptone L37, 0.005 g yeast extract, 0.1 g lactic acid, 0.4 g citric Acid, 2.1 g NaHCO3, 10 g urea, 0.07 g uric acid, 0.8 g creatinine, 0.37 g CaCI2-2H20, 5.2 g NaCI, 0.0012 g FeSO4-7H2O, 3.2 g Na₂O₄S ■ 10 H₂O, 0.95 g KH2PO4, 1 .2 g HK₂O₄P, 1 .3 g NH4CI were made up to 1 L with distilled water. HCI (molarity e.g. 2.5 M) was used to adjust the pH to 6.5. Finally, the medium was filter-sterilized via a 0.22 pm membrane-filter (Millipore).

Confocal Laser Scanning Microscopy

Confocal Laser Scanning Microscopy (CLSM) was used to visualize and generate Z-stack representations for the mixed species biofilms. Visualisation was performed with a Zeiss LSM 700 confocal laser scanning microscope (Carl Zeiss, Ltd, Welwyn, UK) controlled by the ZEN 2009 software platform (Carl Zeiss, Ltd, Welwyn, UK), using a 5x or 10x objective.

The acquisition settings depended on the fluorescence: pinhole 1 .0, laser intensity 5%-15% and gain between 450 and 700 for bio-fluorescent strains. Nevertheless, once adjusted to optimize the visualization, settings were never modified within an experiment to facilitate an unbiased comparison. The Z-stack height was set for each acquisition in a new well by starting from the first point where no emission was detected (just prior to the start of the biofilm), through the biofilm itself, until a point of no emission was again reached, which was set as the last slice. Spacing, once set, was kept constant within the experiment.

Biofilm quantification and analysis

Biofilms were analysed and quantified using a TECAN™ infinite F200 PRO plate reader. To enable analysis of Z-stack images generated with CLSM, 3D pictures were processed using the Maximum intensity Projection function in Zen 2.3 SP1 (black) software (Carl Zeiss, Jena, Germany). This tool generates a bi-dimensional (2D) image per channel, constituted by pixels containing the maximum value over all images in the Z-stack at the single pixel location. Each new picture was exported in tiff format (64bit) with no compression into Zen 2.3 (Blue edition). Analysis and quantification were performed on the entire image using the Measure function in ImageJ 1 ,52i software (Rueden et aL, 2017) implemented with Fiji plugin bundle (Schindelin et aL, 2012). The data generated per single picture are the man grey value defined as sum of the grey values of all the pixels, divided by the number of pixels and the related standard deviation. Final data represent the mean values of the samples grown under a specific condition as the average of the mean grey values generated among the collected images per well. The resulting graphic is shown in figures 3 and 4.

Claims

1 . Use of a lactam composition to improve toilet hygiene, the use comprising treatment of a toilet surface, wherein the composition comprises from 0.0001 to 5 wt.% of a lactam; wherein the use involves reduction of malodour; or wherein the use involves reduction of unwanted urine scale formation in the toilet; wherein the lactam is of formula (I) or (II): wherein:

Re is selected from hydrogen and methyl; and

2. Use according to claim 1 , wherein the lactam is present at a level of from 0.0001 to 2.5 wt.%, preferably from 0.0001 to 1 wt.%, more preferably from 0.001 to 1 wt.%.

3. Use according to claim 1 or claim 2, wherein the reduction of malodour involves the reduction of malodour associated with urine, in particular relating to the reduction of malodour associated with phenol and indole.

4. Use according to claim 1 or claim 2, wherein reduction of unwanted urine scale in the toilet involves the inhibition of P. mirabilis.

5. Use according to any preceding claim, wherein in the lactam of formula (I) or (II), Ri,

R4 and R₅ are H; R₃ is H, or (CH₂)_nN⁺(CH₃)3, where n is an integer from 1 to 16, preferably 2 to 8; and R₂ is a phenyl group, or a mono-substituted phenyl group; preferably R₂ is selected from phenyl, 4-fluorophenyl, 2-fluorophenyl, 4-chlorophenyl, 3-chlorophenyl, 4-bromophenyl and 4-methylphenyL

6. Use according to any preceding claim, wherein the lactam is a lactam selected from:

7. Use according to any preceding claim, wherein the lactam is selected from:

8. Use according to any preceding claim, wherein the lactam is: chlorophenyl)-5-methylene-pyrrol-2-one.