Detection method for microbial contaminants in detergents

Abstract

Process for detecting gram-negative bacteria and / or fungi contaminants in a detergent comprising gram-positive bacteria comprising the subsequent steps of: Providing a detergent comprising intentionally added spores of Bacillus sp; wherein the amount of spores is from 1x102 to 1x1014 CFU / g of detergent; and Adding a sample of the detergent to a liquid growth medium, wherein the medium comprises an effective amount of gram-positive bacteria selective growth inhibitor, and wherein the medium comprises an effective amount of disinfectant neutralizer to provide a liquid growth mixture; and Incubating the liquid growth mixture for from 6 hours to 48 hours at a temperature of from 18 to 46 degrees Celsius; and Diluting the growth mixture by from 3- to 100-fold to provide a liquid growth mixture dilution; and Lysing cells present in the liquid growth mixture dilution; and Applying an ATP-dependent bioluminescence assay to determine the amount of gram-negative and / or fungi contaminants in the detergent provided at step a.

Description

Detection method for microbial contaminants in detergentsField of the inventionThe invention relates to a process for detecting gram-negative bacteria and / or fungi contaminants in a detergent comprising intentionally added gram-positive bacteria.Background of the inventionDetergents are used to clean hard and / or soft surfaces. Detergents in general contain surfactants and often a variety of further actives such as builders, chelating agents, enzymes, bleach systems, surface modification polymers, depending on the intended use. Domestic detergents are of particular interest and include dishwashing liquids, laundry detergents, liquid hard surface cleaners, skin cleaners, rinse additives and so on. There has been an increasing demand by consumers for detergents which rely on milder chemistry and contain natural actives. The use of microbes and in particular spore-forming gram-positive bacteria such as Bacillus sp. may provide desired bio-based cleaning benefits. Spores are resistant and can be included as detergent additive.One problem with detergents which contain gram-positive bacteria, and which also generally have become milder over time, is the risk of contamination with undesired micro-organisms. These can include gram-negative bacteria such as Escherichia coli and fungi such as Candida sp. Detergent manufacturing plants therefore require methods to analyze samples of detergents for microbial contaminants. The method is desirably simple, reliable, fast, and can detect the presence of microbial contaminants despite the presence of the desired and intentionally added gram-positive bacteria in the detergent.US 5,843,699 discloses a rapid screening method for the detection and identification of target microorganisms in a sample which may contain both target microorganisms and competing nontarget microorganisms. The method comprises pre-enriching a sample in a growth medium; adding at least one inhibitor of the non-target microorganisms to the growth medium to discourage the growth of non-target microorganisms and to encourage the growth of the target microorganisms. Further incubating the sample in the growth medium including the at least one inhibitor for a predetermined amount of time and subsequently performing biochemical assays specific for identification of the target microorganisms; and detecting the presence of the target microorganisms in the sample. The method of US 5,843,699 aims to avoid plating methods and immune-assay methods as these are time consuming and / or can be complex to perform. Themethod of US 5,843,699 focusses mainly on determination of target organisms in foods and relies on biochemical assays which are fine-tuned to detect a target microorganism (and not general non-gram-positive contaminants).WO2013 / 177227 discloses a method of detecting microorganisms by use of a general ATP- dependent bioluminescence method. Applying this method for the detection of microbial contaminants in detergents containing intentionally added gram-positive bacteria was found to be ineffective even when applying the general selective growth method of US 5,843,699.An objective of the current invention is to provide a rapid detection method of gram-negative and fungal microbial contaminants in detergents which contain intentionally added Bacillus sp.Summary of the inventionOne or more of the above objectives is achieved by a process for detecting gram-negative bacteria and / or fungi contaminants in a detergent comprising gram-positive bacteria comprising the subsequent steps of: a. Providing a detergent comprising intentionally added spores of Bacillus sp; wherein the amount of spores is from 1x102to 1x1014CFU / g of detergent; and b. Adding a sample of the detergent to a liquid growth medium, wherein the medium comprises an effective amount of gram-positive bacteria selective growth inhibitor, and wherein the medium comprises an effective amount of disinfectant neutralizer to provide a liquid growth mixture; and c. Incubating the liquid growth mixture for from 6 hours to 48 hours at a temperature of from 18 to 46 degrees Celsius; and d. Diluting the growth mixture by from 3- to 100-fold to provide a liquid growth mixture dilution; and e. Lysing cells present in the liquid growth mixture dilution; and f. Applying an ATP-dependent bioluminescence assay to determine the amount of gramnegative and / or fungi contaminants in the detergent provided at step a.Detailed description of the inventionDefinitionsWeight percentage (wt.%) is based on the total weight of the relevant composition (e.g. detergent composition, liquid growth medium, liquid growth mixture) unless otherwise indicated or as made clear from the context. It will be appreciated that the total weight amount of ingredients will not exceed 100 wt. %. Whenever an amount or concentration of a component isquantified herein, unless indicated otherwise, the quantified amount or quantified concentration relates to said component per se, even though it may be common practice to add such a component in the form of a solution or of a blend with one or more other ingredients. It is furthermore to be understood that the verb "to comprise" and its conjugations is used in its nonlimiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. Finally, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the elements is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one". Unless otherwise specified all measurements are taken at standard conditions. Whenever a parameter, such as a concentration or a ratio, is said to be less than a certain upper limit it should be understood that in the absence of a specified lower limit the lower limit for said parameter is 0.Detergent compositionThe detergent of the invention preferably is a hand dishwash detergent, a machine dishwash detergent, a dishwash rinse aid, a laundry detergent, a fabric conditioner, rim block or a floor cleaner. Floor cleaners, laundry detergents and rinse aids (including machine dishwash rinse aids and laundry rinse aids) are the more preferred.Detergents suitable for use in the process of the invention should be liquid, water-soluble and / or water-miscible. For example, laundry powder detergents are solid, but readily water-soluble. Detergents suitable for use in the process of the invention are those which provide a transparent liquid when diluted in water at a ratio of detergent : water of from 1 : 10 to 1 : 1000. Preferably however, for ease in use of the process, the neat detergents are liquids, more preferably are neat aqueous liquids. The detergent of the invention is beneficially is a liquid detergent, more preferably an aqueous liquid detergent. Preferably the detergent comprises at least 40 wt.%, more preferably 50 wt.%, even more preferably 60 wt.% water and still even more preferably at least 70 wt.% water.The term “liquid” in the context of this invention denotes that a continuous phase or predominant part of the detergent is liquid and that the composition is flowable at 15 degrees Celsius or higher. Accordingly, the term “liquid” may encompass emulsions, suspensions, and compositions having flowable yet stiffer consistency, known as gels or pastes. The viscosity of the detergent is preferably from 200 to about 10,000 mPa.s at 25 degrees Celsius at a shear rate of 21 sec1. This shear rate is the shear rate that is usually exerted on the liquid whenpoured from a bottle. Pourable liquid detergents preferably have a viscosity of from 200 to 1,500 mPa.s, preferably from 200 to 700 mPa.s.The term ‘transparent’ as used herein refers to the ability of light within the visible spectrum (400 to 700 nm) to pass through a material sample. The transparency can be quantified as the ratio between the light intensity measured after the light has passed through a material sample and the light intensity measured when the material sample has been removed. The ratio can be converted to a percentage Transmittance (multiply the ratio x 100) ranging from 0% (no incoming light having passed through the material sample) to 100% (i.e. no difference in light having passed through with and without material sample). Transmittance can be suitably measured by using a LIV-VIS Spectrometer. Suitable LIV-VIS spectrometers are available from a variety of suppliers, including Thermo Scientific, Perkin Elmer and Shimadzu. As used herein the term ‘transparent’ refers to a Transmittance of at least 10% within the wavelength range of 400 to 700 nm, preferably of at least 20 %, 30 %, 40 %, 50 %, 60%, 70%, 80%, in increasing order of preference, and still even more preferably of at least 90 %. The transparency of a liquid is preferably measured by using a light path-length of 1 cm to standardize the measurement. Transparency of liquid is typically determined by use of a cuvette, wherein the transmittance of the liquid is based on the ratio between a cuvette with the liquid detergent versus an empty cuvette (x100). The suitable cuvette to use is usually indicated in the Supplier instructions of the LIV-VIS spectrometer.The detergent when diluted with water at a ratio of detergentwater of from 1:10 preferably has an average transmittance in the wavelength range of from 400 to 700 nm, based on a path- length of 1cm, of at least 70 %, more preferably of at least 80%.In case the detergent is a liquid detergent it preferably has an average transmittance in the wavelength range of from 400 to 700 nm, based on a path-length of 1cm, of at least 30 %, more preferably of at least 50% and even more preferably of at least 70 %.The detergent may be packaged as a bottle or as a unit-dose product. The detergent can be part of a multi-chamber unit dose which comprises further chambers which may included further liquid or non-liquid formulations.Bacillus SporesThe detergent provided at step a) of the process comprises intentionally adding spores ofBacillus sp. in an amount capable of providing a cleaning benefit upon use or after uses of thedetergent. Said cleaning benefit can include malodor reduction for an extended time after cleaning, especially when worn.Preferably, the method of the invention requires the intentional addition of 1x10xCFU / g to the detergent where x is from 2 to 14, more preferably from 3 to 10 and even more preferably from 4 to 9. The CFU count is advantageously measured directly after addition and mixing of the spores with the detergent.Spores play critical roles in long term survival because they are highly resistant to extreme environmental conditions. Spores can remain metabolically dormant for years until germination is triggered by favorable environmental conditions. Such conditions include dilution of the detergent when forming a wash liquor and / or deposition on a surface where they may contact moisture and (soil)nutrients deposited on the surface after cleaning via daily use.Methods for obtaining bacterial spores from vegetative cells are well known in the art. For example, vegetative cells are grown in liquid medium and beginning in the late logarithmic or early stationary growth phase, the bacteria may begin to sporulate. When the bacteria have sporulated, the spores may be obtained from the medium such as by centrifugation. Optionally the spores may be purified by (partial) removal of remaining vegetative cells, other cellular debris and / or growth substances. Bacterial spores can be differentiated from vegetative cells using a variety of techniques, like phase-contrast microscopy, automated scanning microscopy, high resolution atomic force microscopy or tolerance to heat, for example. Since bacterial spores are generally environmentally tolerant and are essentially metabolically inert or dormant, they can be readily included in detergent products. Spores can rapidly respond to changing environmental conditions and break dormancy by germinating into a vegetative bacterium.Bacillus sp. are generally spore-forming bacteria and such spores can be used in the detergent. The intentionally added spores of Bacillus sp. preferably include those derived from one or more of Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis,Bacillus megaterium, Bacillus pumilus, Bacillus cereus, Bacillus thuringiensis, Bacillus mycoides, Bacillus tequilensis, Bacillus vallismortis and, Bacillus mojavensis', and more preferably include those derived from one or more of Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus megaterium and Bacillus pumilus.The bacterial spores for use herein are advantageously capable of i) surviving the temperatures found in a laundry dryer; ii) are fabric substantive; iii) have the ability to control odor; and iv)preferably have the ability to germinate during or after the cleaning action to form vegetative cells.SurfactantThe detergent of the invention comprises from 0.5 to 60 wt. % of a surfactant, preferably from 2 to 50 wt.%, more preferably from 5 to 40 wt.% and still more preferably from 10 to 30 wt. % of surfactant. Preferably greater than 95% of the surfactant is selected from anionic and nonionic surfactant and mixtures thereof.In general, the surfactant mixture used can depend on the intended use of the detergent.In general, the anionic surfactants and nonionic surfactants of a surfactant system may be chosen from the surfactants described in Liquid Detergent Detergents edited by E. Smulders (Wiley VCH 2002), Anionic Surfactants: Organic Chemistry edited by Helmut W. Stache (Marcel Dekker 1995), Surfactant Science Series published by CRC press and Non-ionic Surfactants: Organic Chemistry edited by Nico M. van Os (Marcel Dekker 1998), Surfactant Science Series published by CRC press.The preferred amount of total non-ionic surfactant is from 0.1 to 25 wt. %, more preferably from 2 to 20 wt. %, even more preferably from 4 to 16 wt.% and still even more preferably is from 5 to 12 wt.%. Suitable nonionic surfactants may include, in particular, the reaction products of compounds having a hydrophobic group and a reactive hydrogen atom, for example, aliphatic alcohols, acids, amides with alkylene oxides, especially ethylene oxide either alone or with propylene oxide. Specific nonionic surfactants are the condensation products of aliphatic Cs to Cis primary or secondary linear or branched alcohols with ethylene oxide, generally 5 to 40 EO, preferably 7EO to 9EO. For the non-ionic with C16 / 18 alcohol ethoxylates and C16 / 18 Methyl ester ethoxylates it is preferred that the predominant C18 moiety is C18:1 and more preferably C18:1(A9). Preferably polyunsaturated chains are present at less than 11wt%.Preferably the detergent of the invention comprises anionic surfactant in an amount of 2 to 50 wt.%, more preferably from 5 to 40 wt. % and even more preferably from 6 to 30 wt.%.CosurfactantsA detergent of the invention may contain one or more cosurfactants (such as amphoteric (zwitterionic) and / or cationic surfactants) in addition to the non-soap anionic and / or nonionic detersive surfactants described above. Specific cationic surfactants include C8 to C18 alkyldimethyl ammonium halides and derivatives thereof in which one or two hydroxyethyl groups replace one or two of the methyl groups, and mixtures thereof. Cationic surfactant, when included, may be present in an amount ranging from 0.1 to 5 wt.%. Specific amphoteric (zwitterionic) surfactants include alkyl amine oxides, alkyl betaines, alkyl amidopropyl betaines, alkyl sulfobetaines (sultaines), alkyl glycinates, alkyl carboxyglycinates, alkyl amphoacetates, alkyl amphopropionates, alkylamphoglycinates, alkyl amidopropyl hydroxysultaines, acyl taurates and acyl glutamates, having alkyl radicals containing from about 8 to about 22 carbon atoms preferably selected from C12:0, C14:0, C16:0 ,C18:0 and C18: 1 , the term “alkyl” being used to include the alkyl portion of higher acyl radicals. Amphoteric (zwitterionic) surfactant, when included, may be present in an amount ranging from 0.1 to 5 wt.%. Mixtures of any of the above-described materials may also be used.PerfumeThe detergent of invention preferably comprises one or more perfumes. Perfume may be present in the range from 0.1 to 1 wt. %. Many suitable examples of perfumes are provided in the CTFA (Cosmetic, Toiletry and Fragrance Association) 1992 International Buyers Guide, published by CFTA Publications and OPD 1993 Chemicals Buyers Directory 80th Annual Edition, published by Schnell Publishing Co. In perfume mixtures preferably 15 to 25 wt. % are top notes. Top notes are defined by Poucher (Journal of the Society of Cosmetic Chemists 6(2):80

[1955] ). Preferred top-notes are selected from citrus oils, linalool, linalyl acetate, lavender, dihydromyrcenol, rose oxide and cis-3-hexanol.Preferably the perfume contains a substituted benzaldehyde where the substitution is preferably at the at the 3,4 or 5 position, more preferably the subsituent is an alkoxy group. Preferably the benzaldehyde is selected from 4-Methoxybenzaldehyde, 5- Methoxybenzaldehyde, 3- Methoxybenzaldehyde. Preferablythe benzaldhyde is present at from 0.5 to 5wt% of the perfume.Particularly preferred perfume components are selected from verdyl acetate; verdyl propionate; or tert butyl cyclohexyl acetate; tetrahydro linalool; bornyl acetate; bornyl propionate; dihydromyrcenol; hexyl salicylate, Tetramethyl acetyloctahydronaphthalenes; eucalyptol; 2- methyl undecanal; citronellol; geraniol; benzyl acetate; (E)-4-methyldec-3-en-5-ol.Preferably the perfume contains at least 6 of these components, more preferably each at a level of at least 0.01wt% in the detergent product.Tricyclodecenyl based perfumes are verdyl acetate; verdyl propionate; bornyl acetate and bornyl propionate. The sum of the weights of verdyl acetate; verdyl propionate; bornyl acetate and bornyl propionate, will be called Tricyclodecenyl sum. Tert butyl cyclohexyl acetate is preferably ortho tert butyl cyclohexyl acetate or para tert butyl cyclohexyl acetate.Preferably the perfume contains at least 4, more preferably at least 5 components selected from a tricyclodecenyl perfume; tert butyl cyclohexyl acetate; dihydromyrcenol; Tetramethyl acetyloctahydronaphthalenes; hexyl salicylate; tetrahydrolinalool.Preferably the weight ratio of Tricyclodecenyl sum: tert butyl cyclohexyl acetate: dihydromyrcenol is selected from 30 to 40 : 20 to 30: 10 to 20 for long lasting perfumes and 5 to 15 : 20 to 30 : 35 to 50 for freshness perfumes.Preferably the weight ratio of Tetramethyl acetyloctahydronaphthalenes :hexyl salicylate is from 5 to 15 : 10 to 16. Preferably the weight fraction of dihydromyrcenol / hexyl salicylate is from 0.5 to 6.0, more preferably from 1.0 to 3.0.Builders and sequestrantsThe detergent of the invention preferably contains organic detergent builder or sequestrant material. Examples include the alkali metal, citrates, succinates, malonates, carboxymethyl succinates, carboxylates, polycarboxylates and polyacetyl carboxylates. Specific examples include sodium, potassium and lithium salts of oxydisuccinic acid, mellitic acid, benzene polycarboxylic acids, and citric acid. Other examples are DEQUEST™, organic phosphonate type sequestering agents sold by Monsanto and alkanehydroxy phosphonates.Other suitable organic builders include the higher molecular weight polymers and copolymers known to have builder properties. For example, such materials include appropriate polyacrylic acid, polymaleic acid, and polyacrylic / polymaleic acid copolymers and their salts, for example those sold by BASF under the name SOKALAN™. If utilized, the organic builder materials may comprise from about 0.5 to 20 wt. %, preferably from 1 to 10 wt. % percent, of the detergent. The preferred builder level is less than 10 wt. % and preferably less than 5 wt. % percent of the detergent. More preferably the detergent is a non-phosphate built detergent formulation, i.e. , contains less than 1 wt.% of phosphate. Most preferably the detergent formulation is not built meaning it contains less than 1 wt. % of builder. A preferred sequestrant is HEDP (1 - Hydroxyethylidene -1 ,1 ,-diphosphonic acid), for example sold as Dequest 2010. Also suitable but less preferred, as it gives inferior cleaning results, is Dequest(R) 2066 (Diethylenetriamine penta(methylene phosphonic acid or Heptasodium DTPMP).It is preferred, in view of reducing environmental eutrophication problems that the detergent comprises less than 1 wt. % of phosphate, phosphonate or a mixture thereof, more preferably it contains less than 0.8 wt. %, even more preferably less than 0.5 wt. % and still even more preferably less than 0.15 wt. % of phosphate, phosphonate or a mixture thereof.The following builders are especially preferred: 2 ,2',2"-nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTPA), iminodisuccinic acid (IDS), ethylenediamine-N,N'-disuccinic acid (EDDS), methylglycine-N,N- diacetic acid (MGDA), glutamic acid-N,N-diacetic acid (GLDA), N-(2-hydroxyethyl)iminodiacetic acid (EDG), aspartic acid-N-monoacetic acid (ASMA), aspartic acid-N,N-diacetic acid (ASDA), aspartic acid-N-monopropionic acid (ASMP), iminodisuccinic acid (IDA), N-(sulfomethyl)aspartic acid (SMAS), N-(2-sulfoethyl)-aspartic acid (SEAS), N-(sulfomethylglutamic acid (SMGL), N-(2- sulfoethyl)-glutamic acid (SEGL), N-methyliminodiacetic acid (MID A), serine-N,N-diacetic acid (SEDA), isoserine-N,N-diacetic acid (ISDA), phenylalanine-N,N-diacetic acid (PHDA), anthranilic acid-N,N-diacetic acid (ANDA), sulfanilic acid-N,N-diacetic acid (SLDA) , taurine- N,N-diacetic acid (TLIDA) and N'-(2-hydroxyethyl)ethylenediamine-N,N,N'-triacetic acid (HEDTA), diethanolglycine (DEG). Although these builder’ species are mentioned using their acid form, it is to be understood that their partial or full salt forms are included in this denomination. The acid forms of the builder’ are preferred. These builders are preferably present in an amount of from 0.01 to 10 wt. %, more preferably from 0.2 to 5 wt. %.In particular, for machine dishwash detergents it is beneficial if these contain MGDA, GLDA, citric acid or a combination thereof.One or more enzymes are preferably present in the detergent of the invention. Preferably the level of each enzyme in the detergent is from 0.0001 wt. % to 0.1 wt. % protein. Amounts of wt. % enzymes in the detergent refer to wt. % of active protein levels, unless otherwise indicated.Preferably, the detergent may comprise an effective amount of one or more lipases, cellulases, proteases, amylases, hemicellulases, peroxidases, hemicellulases, xylanases, xantanase, lipases, phospholipases, esterases, cutinases, pectinases, carrageenases, pectate lyases, keratinases, reductases, oxidases, phenoloxidases, lipoxygenases, ligninases, pullulanases, tannases, pentosanases, malanases, p-glucanases, arabinosidases, hyaluronidase,chondroitinase, laccase, tannases, nucleases (such as deoxyribonuclease and / or ribonuclease), phosphodiesterases, or mixtures thereof.Examples of preferred enzymes are sold under the following trade names Purafect Prime®, Purafect®, Preferenz® (DuPont), Savinase®, Pectawash®, Mannaway®, Lipex ®, Lipoclean ®, Whitzyme ® Stainzyme®, Stainzyme Plus®, Natalase ®, Mannaway ®, Amplify ® Xpect ®, Pristine®, Celluclean ® (Novozymes), Biotouch (AB Enzymes), Lavergy ® (BASF).Detergent enzymes are discussed in W02020 / 186028(Procter and Gamble), W02020 / 200600 (Henkel), W02020 / 070249 (Novozymes), W02021 / 001244 (BASF) and WO2020 / 259949 (Unilever).A nuclease enzyme is an enzyme capable of cleaving the phosphodiester bonds between the nucleotide sub-units of nucleic acids and is preferably a deoxyribonuclease or ribonuclease enzyme. Preferably the nuclease enzyme is a deoxyribonuclease, preferably selected from any of the classes E.C. 3.1.21.x, where x=l, 2, 3, 4, 5, 6, 7, 8 or 9, E.C. 3.1.22.y where y=l, 2, 4 or 5, E.C. 3.1.30.Z where z= 1 or 2, E.C. 3.1.31.1 and mixtures thereof.PreservativeFood preservatives are discussed In Food Chemistry (Belitz H.-D., Grosch W., Schieberle), 4th edition Springer. Isothiazolone based preservative may also be used. The detergent preferably contains a preservative or a mixture of preservatives, selected from benzoic acid and salts thereof, alkylesters of p-hydroxybenzoic acid and salts thereof, sorbic acid, diethyl pyrocarbonate, dimethyl pyrocarbonate, preferably benzoic acid and salts thereof, most preferably sodium benzoate. The preservative is preferably present at 0.1 to 3 wt.%, preferably 0.3 wt.% to 1.5 wt.%, where the weight is calculated for the protonated form of the preservative.HydrotropesA detergent of the invention may incorporate non-aqueous carriers such as hydrotropes, cosolvents and phase stabilizers. Such materials are typically low molecular weight, water-soluble or water-miscible organic liquids such as C1 to C5 monohydric alcohols (such as ethanol and n- or i-propanol); 02 to C6 diols (such as monopropylene glycol and dipropylene glycol); 03 to 09 triols (such as glycerol); polyethylene glycols having a weight average molecular weight (Mw) ranging from about 200 to 600; C1 to 03 alkanolamines such as mono-, di- and triethanolamines; and alkyl aryl sulfonates having up to 3 carbon atoms in the lower alkyl group (such as the sodium and potassium xylene, toluene, ethylbenzene and isopropyl benzene(cumene) sulfonates). Mixtures of any of the above-described materials may also be used. Nonaqueous carriers, when included, may be present in an amount ranging from 0.1 to 20 wt.%, preferably from 2 to 15 wt.%, and more preferably from 10 to 14 wt.%. The level of hydrotrope used is linked to the level of surfactant and it is desirable to use hydrotrope level to manage the viscosity in such detergents. The preferred hydrotropes are monopropylene glycol and glycerol. Preferably the detergent contains less than 2 wt.% ethanol, more preferably less than 0.5 wt.% ethanol, preferably it is essentially devoid of ethanol.External StructurantsDetergents of the invention may have their rheology further modified by use of one or more external structurants which form a structuring network within the detergent. Examples of such materials include hydrogenated castor oil, microfibrous cellulose and citrus pulp fibre. The presence of an external structurant may provide shear thinning rheology and may also enable materials such as encapsulates and visual cues to be suspended stably in a liquid.PHThe suitable pH of the detergent depends on the type of detergent. Preferably the detergent has a pH from 2 to 13, preferably from 3 to 11 , as measured at 293K in case it is a laundry detergent. In case the detergent is a hard-surface cleaner or a rinse aid, the pH is preferably from 2 to 7.0, more preferably from 2.5 to 6.0. For laundry detergents the pH is preferably from 7.5 to 10, more preferably from 8 to 9.5. If the detergent is non-liquid, these preferred pH ranges apply to a 10-fold dilution of the detergent in water.Further IngredientsThe detergent may comprise further ingredients such as bleach, bleach activator, bleach catalyst, suds suppressor, polymeric thickeners, soil release polymers and others as desired. Preferably soil release polymers will typically be incorporated into the detergent in concentrations ranging from 0.01 to 10 wt.%, more preferably from 0.1 to 5 wt.%.Preferably polymeric thickeners will typically be incorporated into the detergent in concentrations ranging from 0.01 to 5 wt.%, more preferably from 0.1 to 3 wt.%.Laundry rinse aids preferably comprise 0.5 to 20 wt.% of fabric softening active in particular quaternary ammonium compound as wells as anionic microcapsules encapsulating perfume.ProcessAt step a. of the process of the invention a detergent comprising intentionally added spores ofBacillus sp. is provided. The detergent can be prepared by ordinary means known in the add. Inits simplest form this involves simple adding and mixing the ingredients. Beneficially the spores are added as (one of) the last ingredients of the formulation.The detergent, if it is a liquid, is preferably packaged in a bottle or as liquid phase of a multiphase unit-dose product, such as a multi-chamber water-soluble unit dose.Samples of the detergent are analyzed for the presence of gram-negative bacteria and fungal contaminants.At step b. of the process a sample of the detergent is added to a liquid growth medium. Liquid growth medium is chosen which can support the growth of Escherichia coli and Candida albicans as prime model contaminant organisms. Such media are known in the art (Bonnet et. al. Bacterial culture through selective and non-selective conditions: the evolution of culture media in clinical microbiology. New Microbes New Infect 2020 Mar; 34: 100622). Such media are usually aqueous which contain suitable amounts of nutrients and a neutral pH (usually from 5 to 8 as measure at 25 Degrees Celsius). Suitable media include a source carbon, nitrogen, trace elements (including minerals and vitamins) and optional growth factors.The amount of detergent sample added to the liquid growth media is optimized to balance representative sampling of the detergent, the detection limits of the bioluminescence assay and the time needed for incubation. It was found that preferred effective dilutions of detergent sample: liquid growth medium is from 1 :1000 to 1 :40, more preferably from 1 :600 to 1:50, even more preferably from 1 :400 to 1 :60 still even more preferably from 1:250 to 1 :70 and still more preferably from 1 :150 to 1:80.Preferred media contain from 0.5 to 5 wt.% of peptone, casein hydrolysate, malt extract, meat extract, and yeast extract or a combination thereof. Preferred media also contain some added sodium chloride (at a level of from 0.1 to 1 wt.%).The liquid growth medium also contains an effective amount of disinfectant neutralizer. The requires neutralizers depend on the detergent formulation. Examples of disinfectant ingredients and suitable neutralizers can be found in the table below:It was found that Letheen broth, TAT broth and D / E Neutralizing broth are suitable commercially available growth media which include effective amounts of disinfectant neutralizers to allow cultivation of gram-negative bacteria and fungal microbes if present. It was found that both Letheen and TAT broth allowed growth of Escherichia coli (when spiked in the detergent) when a 1 ml sample of detergent was added to 100 ml of the broth.The liquid growth media preferably contains from 0.01 to 2 wt. %, preferably from 0.02 to 1 wt. % and even more preferably from 0.04 to 0.1 wt.% of lecithin.The liquid growth media preferably contains from 0.05 to 5 wt. %, preferably from 0.1 to 2 wt. % and even more preferably from 0.2 to 1 wt.% of polysorbate. Preferred polysorbates are polysorbate 20, 40, 60 and / or 80, more preferred are polysorbate 60 and / or 80 and even more preferred is polysorbate 80.The liquid growth media also contains an effective amount of an agent which selectively inhibits the growth of gram-positive bacteria (as compared to the growth of gram-negative bacteria and fungal microbes). Such selective gram-positive growth inhibiting agents include Penicillin’s and semisynthetic Penicillin e.g. Amoxycillin, Vancomycin, Acriflavines, sodium citrate, bile salts, ox gall, potassium tellurite, lauryl sulphates, methylene blue, eosin, crystal violet and / or ethyl violet (Bonnet et. al. Bacterial culture through selective and non-selective conditions: the evolution of culture media in clinical microbiology. New Microbes New Infect 2020 Mar; 34: 100622).It was surprisingly found that selective growth inhibitors which are dyes may interfere with some types of bioluminescence detection methods. Preferred selective growth inhibitors are Penicillin’s and semisynthetic Penicillin e.g. Amoxycillin, Vancomycin, Acriflavines, sodium citrate, bile salts, ox gall, potassium tellurite and / or lauryl sulphates and more preferred are sodium citrate, bile salts, ox gall, potassium tellurite and / or lauryl sulphates.It was surprisingly found that the amount used of such selective gram-positive inhibitors is important. Too little and the growth of gram-positive bacteria may not be sufficiently suppressed. Too much and the growth of gram-negative and fungal contaminants may be inhibited as well. This is not trivial since the process of the invention is directed to fast detection of microbial contaminants. Hence if the liquid growth medium with disinfectant neutralizer and gram-positive inhibiting agent slows down the out-growth of potential contaminants too much than the process may lead to false negatives.A particularly effective amount of selective growth inhibitor was found to be from 0.5 to 5 wt.%, preferably 0.7 to 4 wt.%, even more preferably from 0.8 to 3 wt.% and still even more preferably from 1 to 2.5 wt.%. such more preferred levels strike an optimal balance between inhibiting growth of gram-positive bacteria (in particular Bacillus sp.), while little affecting the growth of gram-negative bacteria (in particular Escherichia coli) and fungal microbes (in particular Candida albicans). The applies specially to used amounts of bile salts and ox gall. The more preferred gram-positive growth inhibitor was found to be taurocholate and even more preferably when used at an amount of from 1 to 3 wt.%.In view of the above, the preferred growth mixture used at step c has the following composition:• from 0.5 to 5 wt.% of peptone, casein hydrolysate, malt extract, meat extract, and yeast extract or a combination thereof; and• from 0.04 to 1 wt. % of lecithin; and• from 0.2 to 1 wt.% of polysorbate 80; and• from 0.8 to 3 wt.% of bile salts; and• water to balance.At step c. the liquid growth mixture is incubated at suitable conditions to allow growth of gramnegative and fungal microbe contaminants to reach detection levels of the bioluminescence assay. It was observed that for Escherichia coli and Candida albicans optimal temperature conditions were from 18 to 46 degrees Celsius, more preferably from 24 to 40 degrees Celsius, even more preferably from 25 to 38 degrees Celsius and even more preferably from 26 to 34 degrees Celsius.To improve growth conditions during the incubation, the growth conditions are done under orbital agitation. Advantageous culture conditions are under orbital agitation at from 50 to 400 rpm, more preferably from 75 to 300 rpm and even more preferably from 100 to 200 rpm. Flasksare advantageously used as incubation vessel with a fill volume of 5 to 40 vol.%, preferably 10 to 25 vol.% of the flask volume. Suitable shake flasks are commercially available.Optimal incubation times are from 8 to 48 hours, more preferably from 12 to 40 hours, even more preferably from 18 to 35 hours, still even more preferably from 20 to 28 hours and still even more preferably from 21 to 25 hours.In view of the above a preferred incubation condition at step c. of the process involves:• an incubation temperature of from 25 to 34 degrees Celsius; and• for a time of from 18 to 28 hours, more preferably from 21 to 25 hours; and• under orbital agitation at 75 to 300 rpm; and• in a flask having a fill volume of from 10 to 25 vol. %.At step d. of the process the growth mixture is diluted with a post-enrichment solution at a ratio growth mixture : post-enrichment solution of from 1:2 to 1:100 preferably 1 :3 to 1:100. Such a further dilution was found to be required to improve the detection sensitivity of the bioluminescence assay to detect gram-negative and fungal microbial contaminants. More preferred dilution ratios are from 1:4 to 1:70, even more preferably from 1:6 to 1 :50, still even more preferably from 1 :8 to 1 :30 still even more preferably from 1 :9 to 1:20, and still even more preferably from 1 : 10 to 1 : 15.The dilution is preferably accomplished by adding further liquid growth medium and / or further water, for the sake of convenience. The dilution may also be accomplished by using another aqueous liquid to reduce the level of detergent ingredients, which are carried over from the detergent. Any suitable liquid, which of course does not itself significantly interferes with steps e. and f. can be used to dilute the growth mixture. This can be easily established by use of negative and positive control samples.Lysing cells present in the liquid growth mixture dilutionFor effective ATP-dependent bioluminescence to detect micro-organisms in the postenrichment dilution mixture, a cell-lysis step is required to expose marker molecules. Cell lysis can be accomplished by a variety of methods, such as one or more mechanical and / or chemical processes, but is preferably performed by chemical processes. In such chemical processes the cells are exposed to a cell-lysing agent to release marker molecules / marker enzymes from the cells. The cell marker molecules preferably are one or more of adenylate kinase (AK), adenosine diphosphate (ADP) and adenosine triphosphate (ATP).Preferably marker molecules are amplified to enhance the bioluminescence detection sensitivity. In this case, the cells are preferably subjected to the combination of cell-lysing agent and excess substrate which lyses the cells and amplifies the cell contents. Incubation with cell lysing agent in conjunction with excess substrate is preferably conducted at room temperature between from 15 to 120 min, more preferably from 30 to 90 min and even more preferably from 50 to 80 min.Although mechanical lysis techniques and lysis reagents that only destroy the cell membranes are useful, chemical lysis reagents are preferred. Lysis techniques and reagents such as detergents commonly used in the field may also be useful in the present methods. Non-limiting examples of extractants / lysis agents include detergents (e.g., cationic, nonionic and zwitterionic detergents, such as, CHAPS and the Triton-X series of non-ionic detergents) and antibiotics, and these agents can be commonly used for these lysis purposes. The lysed cell contents, including the marker molecules, are preferably separated from any non-lysed cells and cellular debris. This may be accomplished by filtering of centrifugation and capturing the filtrate or supernatant. Suitable filters are commercially available, for example from M ILLI PORE.Applying an ATP-dependent bioluminescence assayATP-dependent bioluminescence assays were found to be especially advantageous to measure microbial contaminants in detergent products. It was found that ATP bioluminescence assays provided objective, sensitive and quantifiable results.ATP-dependent bioluminescence assays involve the production of "amplified" ATP by using an adenylate kinase (AK)-catalyzed reaction as follows:2 ADP AMP + ATP.The reaction utilizes adenylate kinase enzyme released from the cells by lysis and adding to the lysed cells excess ADP substrate (which is the limiting substrate) to provide more readily detectable levels of ATP. This reaction can increase the native levels of ATP found in cells rapidly by a factor 1000. The preferred reaction time of from 5 to 60 min, more preferably from 10 to 45 min and even more preferably from 15 to 30 min. The incubation temperature of preferably from 20 to 40, more preferably from 22 to 35, even more preferably from 25 to 30 degrees Celsius.The ATP formed can subsequently be measured using a detection assay reagent, (e.g., luciferin / luciferase typically further including Mg2+) and measured based on the luciferase reaction as follows:ATP + Luciferin + O2 — > Amp + PPi (inorganic pyrophosphate) + Oxyluciferin + CO2 + Light.Measured light emission can thus indirectly indicate the presence of microorganisms. A preferred light detection device is a luminometer.The sensitivity of the detection can be determined by using positive control samples of detergent which have been spiked with known amounts of cells which are considered contaminants (e.g. Escherichia coli and Candida albicans). In a similar way the method can be also become quantitative.A positive result may be determined by using any statistically valid interpretive method. Preferably if the detected amount of light of a test sample is at least two times, more preferably at least 5 times, even more preferably at least 10 times, even more preferably at least 20 times the amount of light emitted by a control sample (known to be free from microbial contaminants), then a positive result of contamination is likely present. Providing a control sample known to be free from microbial contaminant can be performed in laboratory conditions using known techniques. Such a laboratory control sample advantageously is re-measured when changes are made to the detergent formulation or the detection process.The process of the invention has been validated for a number of microbial contaminant species which include Escherichia coli, Pseudomonas aeruginosa, Pseudomonas gergoviae, Pseudomonas putida and Candida albicans, although it is considered to be able to detect gramnegative and fungi contaminants in general. Preferably the process detects Escherichia coli, Pseudomonas sp. and Candida sp. contaminants, more preferably detects Escherichia coli, Pseudomonas aeruginosa, Pseudomonas gergoviae, Pseudomonas putida and Candida albicans contaminants; and even more preferably detects Escherichia coli, Pseudomonas aeruginosa, Pseudomonas gergoviae, Pseudomonas putida contaminants; and still even more preferably detects Pseudomonas aeruginosa, Pseudomonas gergoviae, Pseudomonas putida contaminants.It was found that the process was especially sensitive to detect Candida sp. Pseudomonas sp. when compared to Escherichia coli. Therefore, preferably the process is for detectingPseudomonas sp. and / or Candida sp. and even more preferably for detecting Pseudomonas sp contaminants. Of the Pseudomonas species the method was especially effective to detect contaminations of Pseudomonas gergoviae and Pseudomonas putida. Hence still even more preferred the process is for detecting Pseudomonas gergoviae and / or Pseudomonas putida contaminants.ExamplesA liquid detergent was made according to the following formulation in Table 1, which comprises spores of Bacillus sp. The formulation was made by mixing the ingredients to homogeneity.Table 1: Detergent formulationExample 1To determine effective levels of taurocholate to allow selective outgrowth of microbial contaminants, Letheen broth was inoculated with Escherichia coli (47 CFU / ml) and incubated at 37 degrees Celsius for 24 hours in a shake-flask in an orbital shaker set at -200 rpm. The outgrowth of the E.coli inoculum was determined using a bioluminescence method as set out above. It was determined that a level of below 3 wt.% taurocholate allowed sufficient outgrowth of E.coli in the growth conditions tested (i.e. leading to a positive test in the bioluminescence method). At higher levels of taurocholate, outgrowth of E.coli was inhibited leading to negative contamination results in the bioluminescence method. A level of around 2 wt. % taurocholate was determined to be optimal for selective outgrowth of E.coli.Table 2. Composition of the Letheen Broth (pH 7.0 at 25 degrees Celsius)Example 2The influence of taurocholate on the bioluminescence test was subsequently investigated.A detergent formulation as set out in Table 1 was incubated in Letheen broth which contained 2 wt.% taurocholate. The detergent formulation was added to the Letheen mixture at a ratio of1 :100. It was verified that 2 wt.% taurocholate is an effective level to prevent outgrowth of Bacillus sp. when incubating at 37 degrees Celsius for 24 hours in an orbital shake flask set at -200 rpm. Surprisingly it was found that including 2 wt.% of taurocholate as selective inhibitory growth agent in some conditions lead to false positive results in the bioluminescence method (when no contaminants were added). The negative influence of taurocholate was avoided by diluting the liquid growth mixture 10-fold in water, before applying the bioluminescence method.Example 3Having determined effective levels of taurocholate and an effective protocol to treat the post- enrichment mixture for bioluminescence analysis, in this Example the method was evaluated using a variety of common microbial contaminants. To this end the liquid detergent of Table 1 was spiked with a variety of microorganisms as contaminants as set out in table 2:Table 2: spiking overview of different microorganisms in the detergent.After spiking according to Table 2, 1 ml of the liquid detergent was added to 100 ml of liquid growth medium. The liquid growth medium used was Letheen Broth + 2 wt.% taurocholate.The mixtures were subsequently incubated at 37 degrees Celsius for 24 hours. The incubation was performed in flasks on an orbital shaking incubator set at 200-250 rpm. The influence of interfering ingredients on the assay is reduced by performing a further dilution. After incubation, the liquid growth mixture was diluted with water at a ratio liquid growth mixture : water of 1 : 10.The diluted samples were analyzed using the Amplified ATP bioluminescence (Celsis AMPiScreen, Supplier Charles River) product-kit. The method is based on amplifying the amount of ATP by use of adenylate kinase and to subsequently measure the total ATP using an ATP dependent bioluminescence protein. Measurement of the bioluminescence signal was done using a Celsis Advance II Luminometer (preferred luminometer of the invention) operated using the standard recommended assay parameters and under control of the Advance. im v 4.0.6 software (preferred software of the invention). The standard recommended assay parameters and the software are supplied with the Luminometer. The results of the Luminometer are obtained as Relative Light Units (RLU). The measured RLU values from the Examples were subtracted from the non-spiked Control (See Table 2).A difference of 3 RLU, preferably 100 RLU more preferably 1000 RLU is used as threshold value. A RLU value above a 1000 RLU’s is considered to be severely contaminated. Although a threshold level of 1000RLU difference is used in this example, as desired the threshold level can be set to a lower (more stringent) or higher (less stringent). A suitable threshold level depends on the acceptable amount of false positive and false negative results, which is turn depends on the number of samples tested. For example, if the chance of a false negative result is 2%, this may be acceptable if 4 samples are tested in parallel, since the chance of a false negative result four times independently will be < 0.0001%.Table 5: Relative Light Units

Claims

Claims1. Process for detecting gram-negative bacteria and / or fungi contaminants in a detergent comprising gram-positive bacteria comprising the subsequent steps of: a. Providing a detergent comprising intentionally added spores of Bacillus sp; wherein the amount of spores is from 1x102to 1x1014CFU / g of detergent; and b. Adding a sample of the detergent to a liquid growth medium, wherein the medium comprises an effective amount of gram-positive bacteria selective growth inhibitor, and wherein the medium comprises an effective amount of disinfectant neutralizer to provide a liquid growth mixture; and c. Incubating the liquid growth mixture for from 6 hours to 48 hours at a temperature of from 18 to 46 degrees Celsius; and d. Diluting the growth mixture by from 3- to 100-fold to provide a liquid growth mixture dilution; and e. Lysing cells present in the liquid growth mixture dilution; and f. Applying an ATP-dependent bioluminescence assay to determine the amount of gramnegative and / or fungi contaminants in the detergent provided at step a.

2. Process according to claim 1 , wherein the detergent comprises an intentional addition of 1x10xCFU / g of detergent, wherein x is from 2 to 14, preferably from 3 to 10 and more preferably from 4 to 9; wherein he CFU count is preferably measured directly after addition and mixing of the spores with the detergent.

3. Process according to claim 1 or claim 2, wherein the added Bacillus sp. spores comprises those derived from one or more of Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus cereus, Bacillus thuringiensis, Bacillus mycoides, Bacillus tequilensis, Bacillus vallismortis and, Bacillus mojavensis', and preferably comprises those derived from one or more of Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus megaterium and Bacillus pumilus.

4. Process according to any preceding claim, wherein the process is for detecting Pseudomonas sp. Candida sp or a combination thereof, and preferably is for detecting Pseudomonas gergoviae, Pseudomonas putida or a combination thereof.

5. Process according to any preceding claim, wherein the detergent comprises from 0.5 to 60 wt. % of a surfactant, preferably from 2 to 50 wt.%, more preferably from 5 to 40 wt.% and still more preferably from 10 to 30 wt. % of surfactant.

6. Process according to any preceding claim, wherein the detergent is a liquid, preferably an aqueous liquid, wherein the aqueous liquid preferably comprises at least 40 wt.%, more preferably 50 wt.%, even more preferably 60 wt.% and still even more preferably at least 70 wt.% water.

7. Process according to any preceding claim, wherein the disinfectant neutralizer comprises from 0.01 to 2 wt. %, preferably from 0.02 to 1 wt. % and even more preferably from 0.04 to 0.1 wt.% of lecithin, as based on the weight of the liquid growth medium.

8. Process according to any preceding claim, wherein the disinfectant neutralizer comprises from 0.05 to 5 wt. %, preferably from 0.1 to 2 wt. % and more preferably from 0.2 to 1 wt.% of polysorbate, as based on the weight of the liquid growth medium, whereof preferred polysorbates are polysorbate 20, 40, 60 and / or 80, more preferred are polysorbate 60 and / or 80 and even more preferred is polysorbate 80.

9. Process according to any preceding claim, wherein the selective growth inhibitor is selected from one or more of Penicillins, Vanomycin, Acriflavin, sodium citrate, bile salts, ox gall, potassium tellurite, lauryl sulphates, methylene blue, eosin, crystal violet and ethyl violet, more preferably wherein the selective growth inhibitor is elected from one or more of Penicillins, Vanomycin, Acriflavin, sodium citrate, bile salts, ox gall, potassium tellurite and lauryl sulphates; more preferably is selected from one or more of sodium citrate, bile salts, ox gall, potassium tellurite and / or lauryl sulphates; even more preferably is selected from one or more of bile salts and ox gall.

10. Process according to any preceding claim, wherein the selective growth inhibitor comprises from 0.5 to 5 wt.%, preferably 0.7 to 4 wt.%, even more preferably from 0.8 to 3 wt.% and still even more preferably from 1 to 2.5 wt.% of bile salts, wherein the preferred bile salt is taurocholate.

11. Process according to any preceding claim, wherein at step c. the incubation time is from 8 to 48 hours, preferably from 12 to 40 hours, more preferably from 18 to 35 hours, even more preferably from 20 to 28 hours and still even more preferably from 21 to 25 hours.

12. Process according to any preceding claim, wherein at step d. the dilution of growth mixture: post-enrichment solution is from 1 :4 to 1:40, preferably from 1:6 to 1 :30, more preferably from 1 :8 to 1:20 and even more preferably from 1:9 to 1 :15.

13. Process according to any preceding claim, wherein at step b. the dilution of detergent sample: liquid growth medium is from 1 :1000 to 1:40, preferably from 1:600 to 1:50, more preferably from 1 :400 to 1:60 even more preferably from 1 :250 to 1:70 and still more preferably from 1:150 to 1 :80.

14. Process according to any preceding claim, wherein the liquid growth medium comprises:• from 0.5 to 5 wt.% of peptone, casein hydrolysate, malt extract, meat extract, and yeast extract or a combination thereof; and• a combination of lecithin and polysorbate 80.

15. Process according to any preceding claim, wherein the bioluminescence method involves ATP amplification by use of an adenylate kinase catalyzed reaction and light detection as produced from a luciferin / luciferase reaction.

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