Biomass determination based on activitiy of enzymes with different PH optima

Abstract

Provided is a method for sequential determination of microorganisms that express enzyme activities having different pH optima. The determination is performed on a single sample which is sequentially subjected to one or more enzyme substrates for these enzyme activities, where the substrates release methylumbelliferone derivatives as a result of the enzyme activity and where the substrates are contacted with the sample at an initial pH followed by a different higher or lower pH. As a result, a measurement of the conversion of substrate at the first pH value and a measurement of the total conversion is used to provide for conversion rates in total and at the different pH values. In turn, these conversion rates are used to calculate microorganism number or concentration. Also provided is a kit or apparatus for implementing the method.

Description

[0001] BIOMASS DETERMINATION BASED ON ACTIVITIY OF ENZYMES WITH DIFFERENT PH OPTIMA

[0002] FIELD OF THE INVENTION

[0003] The invention relates to the field of enzyme activity measurements in samples with a view to determine the presence / quantity of viable microorganisms in a sample. The invention in particular relates to the simultaneous measurement of enzyme activities that are characterized by different pH optima in order to gauge the presence / amount of the microorganisms.

[0004] BACKGROUND OF THE INVENTION

[0005] Numerous methods exist for qualitative or quantitative determination of microorganisms in samples suspected of being contaminated with such microorganisms. Traditional methods for this purpose rely on multiplication via cultivation of material from the samples followed by a detection step. This is in particular useful when aiming at detection of specific microorganisms, since culture conditions may be tailored to favor a limited number of species, which can subsequently be distinguished by use of an appropriate detection system.

[0006] However, methods relying on cultivation suffer the drawback of underestimating the number of microorganisms in the original sample when the aim is to determine the total amount of microorganisms of any species in a sample: since culture conditions inevitably will favor the growth of some microorganisms over other, the end result will or can be completely misleading relative to the true amount of microorganisms present in the original sample.

[0007] For the determination of the total amount of microorganisms, it is hence of relevance to utilize a detection method which is unbiased with respect to species and their varying dependence on growth media and culture conditions.

[0008] WO 2005 / 083109 discloses on such unbiased method, where a sample is subjected to a known degree of concentration by retaining microorganisms on a filter surface by filtrating a known amount of the sample and thereafter the microorganisms are directly detected by measurement of an enzyme activity expressed ubiquitously by microorganisms. In preferred embodiments in WO 2005 / 083109, the activity of a hexosaminidase is determined by measuring the conversion rate exerted by such a filter-concentrated sample on a labelled substrate, which upon cleavage releases a fluorescent moiety, the amount of which can be directly correlated with the amount of microorganisms in the concentrated sample, and in turn this activity can then be converted to a concentration of microorganisms in the original sample by taking into account the volume of sample passed through the filter.

[0009] However, while the enzyme activity measured may be ubiquitous, the enzymes having this activity are not and it may for instance be that a sample contains microorganisms expressing the enzyme activity at different pH values. This means that the pH conditions employed in the tests disclosed in e.g. WO 2005 / 083109 will dictate which enzyme species are detected and in turn their corresponding microorganisms: acidic conditions will favor detection of enzymes having acidic pH optima and alkaline conditions will favor detection of enzymes have alkaline pH optima.

[0010] To overcome this, assays are conventionally run in separate individual samples for each enzyme activity with an analysis protocol adjusted to each individual enzyme activity and its pH optimum, thus requiring that multiple parallel assays are run to determine the true enzyme activity (and thereby microorganism content) in a sample.

[0011] OBJECT OF THE INVENTION

[0012] It is an object of embodiments of the invention to provide a simplified assay for microorganisms based on their enzyme activities, where the assay allows detection of enzyme activities of enzymes having different pH optima so as to avoid the need for running multiple parallel assays.

[0013] SUMMARY OF THE INVENTION

[0014] The present invention is based on findings from experiments obtaining enzyme activity measurements in samples using 4-methylumbelliferone labelled fluorogenic enzyme substrate analogues. It has been found that it is possible to obtain simultaneous measurements of enzyme activities that are characterized by different pH optima.

[0015] In particular, it has been found by the present inventor that a modification of the protocols disclosed in WO 2005 / 083109 provides for a simplified assay capable of detection of activity of enzymes having different pH optima

[0016] According to the present invention, the measurements of two enzyme activities with differential pH optima are conducted on the same sample consecutively in one combined routine - thereby the method provides a result for each enzyme activity as well as a total enzyme activity, as well as a ratio determination. Apart from providing a simplified procedure, the present invention also overcomes problems of sample to sample variations that are inherent in existing protocols using parallel testing.

[0017] So, in a first aspect the present invention relates to a method for qualitative or quantitative determination of microorganisms in a sample, the method comprising the sequential steps of a) contacting the sample or a concentrate thereof with a first composition comprising a substrate for a first enzyme, where said first enzyme is expressed by at least a subset of the microorganisms of interest and where the first composition has a first pH value, which favours activity by the first enzyme over activity of enzymes exhibiting activity optima at higher or lower pH values, b) allowing microorganisms present in the sample or concentrate to convert the substrate for a sufficient time to enable detection of substrate conversion at the first pH value, c) adding a second composition whereby the sample or concentrate thereof is contacted with a resulting mixture of the first and second compositions, where the resulting mixture attains a second, higher or lower pH value, which favours activity by a second enzyme over activity of the first enzyme, wherein the second composition optionally comprises more of the substrate defined in step a or optionally comprises a different substrate for the second enzyme, d) immediately measuring the amount of converted substrate present in the resulting mixture, e) allowing microorganisms present in the sample or concentrate to convert the substrate or different substrate for a sufficient time to enable detection of substrate conversion at the second pH value, and f) measuring the amount of converted substrate present in the resulting mixture, and g) based on the measured values in steps d and f determining the amount of microorganisms responsible for conversion of substrate at the first pH, the amount of microorganisms responsible for conversion of substrate at the second pH, and the total amount of microorganisms responsible for conversion of substrate.

[0018] A second aspect of the invention relates to a kit or apparatus, which comprises i. a first composition having a first pH value and comprising a first enzyme substrate, ii. a second composition having a second, higher or lower pH value than the first composition, and comprising either the first enzyme substrate or a different enzyme substrate or no enzyme substrate, ill. a filter unit, where the filter has a pore size that facilitates retention on the influent filter surface of bacteria and other microorganisms when a liquid is passed through the filter in the filter unit, iv. means for drawing or pumping liquid sample material and the first and second compositions through the filter unit, and wherein each enzyme substrate comprises a moiety, which upon enzymatic action on the substrate(s) exhibits detectable properties to allow detection of converted substrate.

[0019] LEGENDS TO THE FIGURE

[0020] Fig. 1 is a bar graph showing microorganism determinations according to the prior art compared to the determination of the present invention.

[0021] Fig. 2 is a graph showing relationship between duration of substrate conversion by microorganisms Acetobacter acetf) and the emitted fluorescence under acidic pH conditions.

[0022] Fig. 3 shows graphs demonstrating linear relationships between duration of substrate conversion by microorganisms and fluorescence and time under acidic pH conditions.

[0023] A: Saccharomyces cerevisiae.

[0024] B: Lactobacillus plantarum.

[0025] DETAILED DISCLOSURE OF THE INVENTION

[0026] Definitions

[0027] "A microorganism of interest" is any microorganism that might be present in a sample and whose presence it can be or relevance to measure, e.g. because the microorganism is a pollutant or pathogen. As such, microorganisms can e.g. be selected from bacteria, fungi (both yeast and filamentous fungi), and protozoans.

[0028] A "concentrate" of a sample of microorganisms is in the current context a processed sample where the volume of an original sample has been reduced while preserving the microorganisms in the sample. For instance, a sample can be filtrated to obtain such a concentrate.

[0029] A "substrate" is a substance susceptible to conversion by an enzyme. In the present context, particular interesting substrates are those that are subject to cleavage as a consequence of the enzymatic reaction. In this context, substrates can release a moiety, which after its release is detectable meaning that the amount of converted / cleaved substrate can be determined by measuring the increase in the amount or concentration of the released moiety. In preferred embodiments, the released moiety can be distinguished from the moiety when it is present in the substrate. For instance, 4-methylumbelliferone exhibits fluorescence characteristics which are clearly distinguishable from those of a methylumbelliferyl group in a substrate.

[0030] A "liquid sample" is in the present context any sample from a location, where the sample has been brought into liquid form, normally in an aqueous solvent. So, a liquid sample can be a sample collected directly from an aqueous environment but may also be a sample from a gaseous or solid medium which subsequently is admixed with an aqueous solvent.

[0031] The terms "influent side" and "effluent side" of a filter describe the compartments of a filter devise which where liquid enters and leaves the filter, respectively.

[0032] The expression "immediately measuring the amount of converted substrate" denotes a determination of converted substrate after establishment of the resulting mixture at a time point where conversion of substrate under the conditions dictated by the higher pH value does not measurably (or at least significantly) influence the measurement of only the conversion observed as a consequence of the pH value of the first composition. There hence exists a (relatively short) time interval after establishment of the resulting mixture where a measurement of the conversion rate provides an acceptable approximation of the pure conversion rate that can be attributed to the pH conditions prevailing when only the first composition has been in contact with the microorganisms of interest. In practice, this acceptable short interval can be determined accurately by conducting a series of parallel experiments where standard samples of microorganisms are used for a determination of conversion rates in a "composition 1 only" setting and comparing this with a set of measurement using the same standard samples in a setting of the present invention, but with increasing time periods from establishment of the resulting mixture until measuring the amount of converted substrate in step d - as long as no difference in conversion rate can be identified, the measurement in step d has been performed "immediately". "Simultaneous measurement" or "simultaneous determination" in the present context is intended to mean that several enzyme activities are determined on the same sample specimen, but the expression does not require that the corresponding measurement are carried out at the same time; typically, the measurements are carried out sequentially, but later measurements are carried out on a sample specimen on which a previous measurement has been carried out.

[0033] Specific embodiments of the invention

[0034] 1staspect of the invention and embodiments thereof

[0035] The first aspect of the invention relates to a method for quantitative or qualitative determination of microorganisms of interest in a liquid sample comprising the steps of a) contacting the sample or a concentrate thereof with a first composition comprising a substrate for a first enzyme, where said first enzyme is expressed by at least a subset of the microorganisms of interest and where the first composition has a first pH value, which favours activity by the first enzyme over activity of enzymes exhibiting activity optima at higher or lower pH values, b) allowing microorganisms present in the sample or concentrate to convert the substrate for a sufficient time to enable detection of substrate conversion at the first pH value, c) adding a second composition whereby the sample or concentrate thereof is contacted with a resulting mixture of the first and second compositions, where the resulting mixture attains a second, higher or lower pH value, which favours activity by a second enzyme over activity of the first enzyme, wherein the second composition optionally comprises more of the substrate defined in step a or optionally comprises a different substrate for the second enzyme, d) immediately measuring the amount of converted substrate present in the resulting mixture, e) allowing microorganisms present in the sample or concentrate to convert the substrate or different substrate for a sufficient time to enable detection of substrate conversion at the second pH value, and f) measuring the amount of converted substrate present in the resulting mixture, and g) based on the measured values in steps d and f determining the amount of microorganisms responsible for conversion of substrate at the first pH, the amount of microorganisms responsible for conversion of substrate at the second pH, and the total amount of microorganisms responsible for conversion of substrate.

[0036] The invention is based on the finding that the second composition can serve as both a medium, which facilitates detection (typically fluorometric) of released detectable moieties from the substrate, and as a medium, which facilitates the further conversion of either the same or a different substrate under more alkaline or acidic conditions. As noted below, this is in particular interesting when the released detectable moiety from the substrate(s) is 4- methylumbelliferone, which requires an alkaline environment in order to be detectable, meaning that the second composition in that case is more alkaline than the first composition.

[0037] In addition, the change in pH in step c has the advantage that the conversion exerted by the first enzyme is stopped or at least reduced significantly, meaning that further conversion of substrate can be ascribed solely to activity of the second enzyme.

[0038] In preferred embodiments of the invention, the liquid sample is concentrated by passing a defined volume thereof through a filter having an influent and an effluent side and having pore sizes sufficiently small to retain the microorganisms of interest on the influent side of the filter surface. This principle is disclosed in detail in WO 2005 / 083109; in brief by relying on concentration of microorganisms in a sample instead of relying on culture thereof, an highly unbiased measurement of viable microorganism concentration can be obtained - when knowing the initial volume concentrated, the correct concentration of microorganisms in the original sample can be determined by simple means by the formula CSampie=Afiiter / VSampie where Csampie is the concentration of microorganisms in the sample, A er is the measured amount of microorganisms in the filter unit ( / .e. caught on the filter surface on the influent side) and Vsampie is the amount of sample passed through the filter unit. There is hence a simple linear relationship between A er and Vsampie for any given concentration of microorganisms in a sample.

[0039] The use of a filter unit is not essential but highly practical. In principle the present invention can be carried out in one reaction chamber, where a sufficient number of microorganisms are present - so it is for instance also possible to specifically capture microorganisms (e.g. by using a chromatographic approach) and then sequentially adding the compositions providing the different pH conditions to the captured microorganisms, optionally after one or more washes. However, when the goal is to capture any microorganisms in a sample the unbiased nature of filtering the sample or using is advantageous.

[0040] When applying a filter as described above, the initial contact between the substrate of the first composition is brought about by adding the first composition to the influent side of the filter to flush out sample liquid from the influent side; the result to attain is that the no appreciable amounts of the original sample liquid remain on the influent side meaning that the addition of the first composition also constitutes a washing step. In common practice of the invention, the influent side of the filter is part of a filter unit or filter device, where the filter itself constitutes at least a part of one of the enclosing walls of an inlet chamber; in this way, the interior part of the filter defines at least one inlet chamber in which the microorganisms that constitute a retentate from the filtration can react with substrate for a time in a closed compartment.

[0041] Likewise, the second composition is added to the influent side of the filter. Again, this can be done by flushing (washing) the inlet chamber with the second composition to force liquid through the filter unit and optionally recycling the filtrate so a thorough mixing of the two compositions is attained. The goal to reach is to allow a measurement of converted substrate and this is typically done in a fraction of the resulting mixture that has been passed to the effluent side of the filter. However, it is in practice also possible to measure the converted substrate directly in the resulting mixture after having mixed the two compositions.

[0042] In order to obtain measurement results that can readily be converted into a microorganism amount, count or concentration, it is advantageous that substrate is always present in nonlimiting amounts to ensure a constant rate of conversion, meaning that the kinetics of the substrate conversion is of first order and only depends on the concentration of the enzyme that acts on the substrate. Preferably, the maintenance of such a sufficient substrate concentration is accomplished by including (sufficient) substrate in each of the compositions. However, in the event the same substrate is utilized at both pH optima, it is possible to instead only include a surplus of substrate in the first composition and then adjust the pH by adding a sufficient volume of the second composition, which in that case can be free of substrate.

[0043] Often, the change in pH in step c facilitates or renders possible the detection of a detectable released moiety. This is in particular true for the mehylumbelliferyl derivatives discussed herein, where the released 4-methylumbelliferone is hardly - if at all - detectable by measuring fluorescence at acidic pH values. However, as such it is not essential that the detection of the released moiety requires a particular pH value. In preferred embodiments of the invention, measuring of substrate conversion comprises determination of fluorescence from a moiety released from substrate as a consequence of the enzyme activity. Again, this is not essential, but measurements of fluorescence are easy to carry out and do not require addition of any reactants to obtain a result as would be the case if the released moiety should be detected by a secondary reaction. Nevertheless, the measurement can instead be of a luminescent moiety, a radioactively labelled moiety, or a moiety exhibiting specific binding to a capture agent. It is in principle also possible to measure a reduction in the amount of non-converted substrate, but this is not preferred due to problems with signal to noise ratios.

[0044] In the preferred embodiments disclosed herein the first pH value is lower than the second pH value - as mentioned above, the methylumbelliferyl derivatives constituting the detectable moiety require alkaline pH to be detected, meaning that the change in pH in step c allows the triple advantage of 1) enabling immediate detection of the substrate converted under the acidic conditions, 2) enabling conversion of substrate by the second enzyme without any further conversion being ascribable to the first enzyme, and 3) finally direct detection of the total amount of converted substrate.

[0045] When measuring of substrate conversion comprises determination of fluorescence from a moiety released from substrate as a consequence of the enzyme activity the substrate(s) is / are as mentioned preferably methylumbelliferyl derivative(s) and the moiety released is 4- methylumbelliferone, which is preferably determined using an excitation wavelength of 365 nm and measured at an emission wavelength of 445 nm.

[0046] When the first pH is lower than the second pH, it is preferred that the first enzyme is an acidic phosphatase and the second enzyme is an alkaline phosphatase. This has the consequence that the substrate can be the same in both steps b and e. Suitable methylumbelliferyl derivative substrates for phosphatases are selected from 6,8-difluoro-4- methylumbelliferyl phosphate, 4-methylumbelliferyl phosphate dicyclohexylammonium salt trihydrate, 4-methylumbelliferyl phosphate free acid; 4-methylumbelliferyl phosphate dilithium salt, and trifluoromethylumbelliferyl phosphate.

[0047] Hence, when concentrating on measurement of a single enzyme activity but at different pH values to favour enzymes having the same substrate specificity but at different pH optima, the conversion of one single substrate is preferably measured. In this case the second composition preferably comprises a further amount of the substrate comprised in the first composition, but as mentioned herein, is also possible that the second composition merely acts by changing pH to an optimum value for another enzyme having the same substrate specificity. It is however possible to utilise substrates being cleaved by enzymes having non-identical substrate specificity in the two compositions. In such a case, the substrate in the first composition can preferably be a substrate for hexosaminidase, such as p-N- acetylhexosaminidase (EC 3.2.52), while the second composition comprises a substrate for a phosphatase. In this case, the substrate in the first composition is typically selected from the group of 4-methylumbelliferyl-p-N-acetyl-D-glucosaminide, 4-methylumbelliferyl-p-D- N,N',N"-triacetylchitotrioside, 5-bromo-6-chloro-3-indolyl-2-acetamido-2-deoxy-p-D-gluco- pyranoside, 5-bromo-4-chloro-3-indolyl-N-acetyl-p-D-glucosaminide, indolyl-2-acetamido-2- deoxy-p-D-gluco-pyranoside, 4-nitrophenyl-N-acetyl-p-D-glucosaminide, p- trifluoromethylumbelliferyl-N-acetyl-p-D-glucosaminide, N-methylum-indolyl-N-acetyl-p-D- glucosaminide, 5-iodo-3-indolyl-N-acetyl-p-D-glucosaminide, 4-methylumbelliferyl-p-D- N,N',N"-triacetylchitotriose, 4-methylumbelliferyl-p-D-N,N'-diacetylchitobioside, 4- methylumbelliferyl-7-(6-sulfo-2-acetamido-2-deoxy)-p-D-glucosaminide, 4- methylumbelliferyl o-D-fucoside; 4-methylumbelliferyl p-D-fucoside, 4-methylumbelliferyl o- D-glucoside, 4-methylumbelliferyl-7-(6-sulfo-2-acetamido-2-deoxy-p-D-glucopyronoside), 4- methylumbelliferyl-N-acetyl-o-D-glucosaminide, 4-methylumbelliferyl p-D-lactoside, 4- methylumbelliferyl-N-acetylgalactosaminide, 4-methylumbelliferyl p-D-mannopyranoside, 4- methylumbelliferyl o-D-mannopyranoside, 4-methylumbelliferyl p-D-xyloside, resorufin-N- acetyl-p-D-glucosaminide, 4-methylumbelliferyl-N-acetyl-o-D-glucosaminide, 9H-(1,3- dichloro-9,9-dimethylacridin-2-one-7-yl)N-acetyl-p-D-glucosa minide (DDAO), and an N- actyl-p-D-glucosaminide oligomer derivative of DDAO preferably 4-methylumbelliferyl-p-N- acetylglucosaminide.

[0048] Again, when using a filter for concentrating the microorganisms, step d) may comprise that a volume of the resulting mixture is passed through the filter from the influent to the effluent side and the amount of the converted substrate is measured in said volume on the effluent side of the filter ( / .e. after passage).

[0049] In order to convert measured substrate conversions to the relevant measure of microorganism activity (count, concentration, amount / mass or other relevant quantity), the measurements are e.g. used for an interpolation against data sets (e.g. standard curves) over known microorganism activities and corresponding substrate conversion measurements performed under the same conditions as in a given practical implementation of the method of the 1staspect. So, as an example, if the practical implementation of the method entails 10 minutes of contact between sample and a substrate under acidic conditions followed by 15 minutes of contact between sample and substrate under alkaline conditions, then the measurement results ascribable to each conversion are interpolated against sets of data obtained under the same 2 sets of conditions for the same 2 periods of time. Hence, in the event the detectable moiety released by the conversion is the same in both steps d and f, then the step d measurement result is ascribable to the acidic condition conversion and the step f measurement result minus step d measurement result is ascribable to the alkaline condition conversion. In the event the released detectable moiety is not the same, then the step f measurement result alone is ascribable to the alkaline condition conversion. In the preferred embodiments herein, the released moiety measured in step d and the released moiety detected in step if are identical, typically 4-methylumbelliferone.

[0050] 2ndaspect of the invention and embodiments thereof

[0051] As mentioned above, the 2ndaspect of the present invention relates to a a kit or apparatus, which comprises i. a first composition having a first pH value and comprising a first enzyme substrate,

[0052] II. a second composition having a second, higher or lower pH value than the first composition, and comprising either the first enzyme substrate or a different enzyme substrate or no enzyme substrate, ill. a filter unit, where the filter has a pore size that facilitates retention on the influent filter surface of bacteria and other microorganisms when a liquid is passed through the filter in the filter unit, iv. means for drawing or pumping liquid sample material and the first and second compositions through the filter unit, and wherein each enzyme substrate comprises a moiety, which upon enzymatic action on the substrate(s) exhibits detectable properties to allow detection of converted substrate.

[0053] When the 2ndaspect is an apparatus, it will typically also comprise a means for detection of a signal from such moieties; as detailed above under the discussion of the first aspect of the invention the means for detection can be a fluorometer adapted to detect fluorescence from the relevant moiety, but the means for detection may also be a detection device for radioactivity, luminescence, or a binding reaction.

[0054] Also, the kit or apparatus is equipped with a filter unit, which typically is so configured that it has a housing defining at least an inlet chamber and preferably an outlet chamber, where the inlet chamber has the filter's influent side facing its interior lumen, and where the inlet chamber is capable of serving as a reaction vessel for conversion of enzyme substrate by microorganisms retained on the influent side of the filter in the inlet chamber. On the other side of the filter, the outlet chamber can accommodate a sufficient volume of liquid to allow determination of the above-discussed moiety - and in certain versions of the apparatus, the detection means is coupled directly to the outlet chamber; for instance, a fluorometer may have its sensor facing or placed in the outlet chamber.

[0055] The means for drawing or pumping the liquid sample can e.g. be constituted by a piston or a other device that exerts an elevated pressure on liquid on the influent side to force it through the filter. Instead, a suction mechanism may decrease the pressure on the effluent side to pull liquid in an equivalent manner across the filter.

[0056] It is preferred that the moity exhibits detectable properties which are optimal or only present at a pH value between the first and second pH values or at a pH value closer to the second pH value than to the first pH value.

[0057] Preferably, the second pH value is higher than the first pH value.

[0058] In the kit or apparatus, each enzyme substrate is a preferably a methylumbelliferyl derivative and the moiety is 4-methylumbelliferone. In this context, all the methylumbelliferyl derivatives discussed above under the discussion of the 1staspect of the invention are possible substrates in the context of the first and second compositions - in this context, the discussion of the components of the first and second compositions apply mutatis mutandis to the discussion of the first and second compositions, respectively, in the kit or apparatus.

[0059] Hence, in the kit or apparatus, a) the first and second composition can comprise the same enzyme substrate; b) the first and second composition may comprise substrates for different enzyme activities; or c) the second composition may not comprise an enzyme substrate. In the case of a or c, the enzyme substrate is a preferably a phosphatase substrate (cf. above under the discussion of the first aspect of the invention for a listing of suitable phosphatase substrates); in the case of b, the enzyme substrate in the first composition is preferably a hexosaminidase substrate, and the substrate in the second composition is preferably a phosphatase substrate.

[0060] EXAMPLE 1

[0061] Combined measurement of microorganisms exhibiting acid and alkaline phosphatase activity

[0062] Preparation of enzyme substrate solutions

[0063] The enzyme substrate solution for determination of acid phosphatase EC 3.1.3.2. (solution ESS 1) was prepared as follows:

[0064] A glycine / NaOH solution (0.03 M glycine, 0.027 M NaOH) was mixed with substrate by dissolving 4-methylumbelliferyl phosphate in the solution to reach a concentration of 0.375 mM. After this, the pH was adjusted to 5.1 with 0.026 M citric acid (pH 2.4). The resulting concentration of 4-methylumbelliferyl phosphate after citric acid addition is 0.125 M.

[0065] The enzyme substrate solution for determination of alkaline phosphatase EC 3.1.3.1 (solution ESS 2) was prepared as follows:

[0066] A glycine / NaOH solution (0.05 M glycine, 0.045 M NaOH) was mixed with substrate by dissolving 4-methylumbelliferyl phosphate in the glycine-NaOH solution to reach a concentration of 0.25 mM. pH was adjusted to 10.0 by addition of 0.026 M citric acid. The resulting concentration of 4-methylumbelliferyl phosphate after citric acid addition is 0.125 M.

[0067] Sampling

[0068] A relevant volume of an environmental liquid water sample suspected of containing microbial contaminants harboring an acid phosphatase and an alkaline phosphatase was collected by transferring the water sample through a closed filter unit with pore size 0.22 pm. The relevant sample volume was recorded as V.

[0069] Analytical blank

[0070] An analytical blank was prepared by transferring 0.35 ml of solution ESS 1 into 2.0 ml of solution ESS 2. Fluorescence was measured at an excitation wavelength of 365 nm and emission wavelength of 445 nm. The resulting fluorescence was recored as "analytical blank". Enzyme activity analysis:

[0071] The enzyme analysis was initiated by transferring 2.5 ml of solution ESS 1 through the closed filter unit. The surplus enzyme substrate solution was discarded. The filter unit was left at 23°C (thermostatically controlled) for reaction for T minutes - the reaction time T is adjusted to the water type to achieve a fluorescence production which is within the linear range with time and sample volume (a depicted in Figs. 1-3). The selected reaction time was recorded as T.

[0072] Intermediate enzyme activity determination (A)

[0073] A fluorescence reading cuvette (10 x 10 x 45 mm) was prepared by transferring 2 ml of solution ESS 2 into the cuvette. When the reaction time (T) had been reached, the filter was flushed two times with solution ESS 2, using a blunt needle attached to the filter luer: This was done by placing the filter with blunt needle in the cuvette and drawing solution ESS 2 from the cuvette up through the filter and back down through the filter, thereby collecting the flushed liquid in the cuvette. This routine was repeated twice. The fluorescence was read on a fluorometer with an excitation wavelength of 365 nm and emission wavelength of 445 nm. The fluorescence reading was recorded as an intermediate fluorescence value and used to calculate the acid phosphatase activity. The acid phosphatase activity is calculated as intermediate relative fluorescence units (RFU) / 250 ml / 30 minutes at 23°C.

[0074] Total enzyme activity determination (B)

[0075] The reaction in the filter was allowed to continue for T minutes and the procedure (A) described above was repeated. The fluorescence reading was registered as fluorescence combined and used to calculate the total enzyme activity, i.e. the acid phosphatase + alkaline phosphatase activity. The enzyme activity is calculated as the total relative fluorescence units generated (RFU) / 250 ml / 30 minutes at 23°C.

[0076] Calculation of contamination:

[0077] Enzyme activity Calculation of enzyme activity used as proxy for contamination level

[0078] Acid Phosphatase (Intermediate fluorescence reading - analytical blank) - adjusted for activity (APase) volume (V) and reaction time (T) Acid Phosphatase (Combined fluorescence reading - analytical blank) - adjusted for activity + alkaline volume (V) and reaction time (T) phosphatase activity (Total enzyme activity)

[0079] Alkaline Total enzyme activity - Acid Phosphatase activity phosphatase (Alkpase)

[0080] Ratio Acid phosphatase activity / Alkaline phosphatase activity

[0081] In another example the acid phosphatase enzyme substrate is substituted with a 4- methylumbelliferone labelled fluorogenic enzyme substrate for hexosaminidase activity EC 3.2.52 and pH optimum of 7.2.

[0082] Discussion:

[0083] The order of the factors is essential for the assay in this example - any changes in the sequence of routines will render the assay useless, due to the pH sensitivity of the fluorophore in the detection step. Surprisingly, it is possible to consecutively use an enzyme substrate solution (exemplified by ESS 2) as both a developer (by making a fluorophore detectable) and at the same time as an enzyme substrate initiating an additional enzyme activity measurement.

[0084] Hygiene test in beverage draft system and test assay validation

[0085] The data in Fig. 1 were obtained from a contaminated draught beer system where there was a suspicion of a predominantly beverage related contamination with acidophilic microorganisms. APase and AlkPase activities were determined on two individual subsamples with specific protocols for each enzyme and the combined assay was performed on one sample.

[0086] The agreement between the individual assays determined in two separate samples and the combined assay determined in one sample is 98.6%

[0087] The data corroborated a predominantly beverage related contamination with acidophilic microorganisms. EXAMPLE 2

[0088] Relative specificity ofAPase and AlkPase in rehydrated freeze-dried cultures of relevant microorganisms.

[0089] Materials and methods

[0090] A rehydration solution was prepared by adding 9 g NaCI / l and 1 g peptone / l to MiliQ water. The rehydration solution was autoclaved prior to use.

[0091] A freeze-dried culture of Acetobacter aceti (strain DSM 3508) was produced in medium containing yeast extract (2 g / l), glucose (2 g / l), and citric acid (1 g / l) with pH adjusted to 5.5. Freeze additive was added followed by freeze drying. The freeze-dried culture was then added to 2 ml rehydration solution. The rehydrated culture contained 4.2 x 107CFU / ml.

[0092] A commercially available concentrated spray dried culture of yeast strain (Saccharomyces cerevisiae) used for commercial wine production was rehydrated by adding 2 ml rehydration solution and kept at 30-35°C for 20 minutes. The yeast culture was very concentrated so further dilution by adding 0.150 ml into 150 ml dilution water was necessary. The final rehydrated and diluted yeast culture contained 9.0 x 105CFU / ml.

[0093] A concentrated freeze-dried culture of Lactobacillus plantarum (strain DSM 4361) was produced in medium containing yeast extract, grape juice and malic acid. It was concentrated on a centrifuge before addition of freeze additives and freeze drying. Subsequently, the freeze dried culture was added to 2 ml rehydration solution. The culture was very concentrated so further dilution was necessary adding two ml culture into 150 ml dilution water. The re-hydrated culture contained 2.8 x 108CFU / ml.

[0094] A freeze-dried generic tap water culture was produced in 1 liter tap water containing 50 mg Bacteriological peptone and 125 mg glucose, followed by incubation for 24 - 48 hours. Freeze-additive was added followed by freeze-drying. Subsequently, the freeze-dried culture was added 2 to ml rehydration solution. The rehydrated culture contained 2.9 x 105CFU / ml.

[0095] The data in Table 1 shows the relative specificity in solutions ESS 1 (favoring Apase activity) and ESS 2 (favoring Alkpase activity) for acidophilic microorganisms (Acetobacter aceti, Lactobacillus plantarum, and Saccharomyces cerevisiae'). Each sample was analyzed for Apse and AlkPase. Freeze dried or spray dried cultures were used. For each sample the highest enzyme activity was set at 100%. Each sample was tested at the same concentration. The 3 cultures were as follows:

[0096] Acetobacter aceti

[0097] Freeze-dried culture was produced in media containing yeast extract (2 g / l), glucose (2 g / l), citric acid (1 g / l), and pH adjusted to 5.5. Freeze additive was added followed by freeze drying. Rehydration : freeze-dried culture is added 2 ml rehydration solution. The rehydrated culture contains 4.2 x 107CFU / ml.

[0098] Lactobacillus plantarum

[0099] Concentrated freeze-dried culture was produced in media containing yeast extract, grape juice and malic acid. It was concentrated on a centrifuge before addition of freeze additives and freeze-drying. Rehydration: freeze dried culture is added 2 ml rehydration solution. The culture is very concentrated so further dilution by adding the two ml culture into 150 ml dilution water is necessary. This contains 2.8 x 108CFU / ml.

[0100] Saccharomyces cerevisiae

[0101] Concentrated spray dried culture of yeast strain used for wine production. Rehydration: spray dried culture is added 2 ml rehydration solution. The yeast culture is very concentrated so further dilution by adding 0.15 ml into 150 ml dilution water is necessary. This contains 9.0 x 105CFU / ml.

[0102] Table 1

[0103] In Figs. 2-3 are shown the measured fluorescence over time for APase conversion of substrate by the microorganisms set forth in Table 1.

Claims

CLAIMS1. A method for quantitative or qualitative determination of microorganisms of interest in a liquid sample comprising the steps of a) contacting the sample or a concentrate thereof with a first composition comprising a substrate for a first enzyme, where said first enzyme is expressed by at least a subset of the microorganisms of interest and where the first composition has a first pH value, which favours activity by the first enzyme over activity of enzymes exhibiting activity optima at higher or lower pH values, b) allowing microorganisms present in the sample or concentrate to convert the substrate for a sufficient time to enable detection of substrate conversion at the first pH value, c) adding a second composition whereby the sample or concentrate thereof is contacted with a resulting mixture of the first and second compositions, where the resulting mixture attains a second, higher or lower pH value, which favours activity by a second enzyme over activity of the first enzyme, wherein the second composition optionally comprises more of the substrate defined in step a or optionally comprises a different substrate for the second enzyme, d) immediately measuring the amount of converted substrate present in the resulting mixture, e) allowing microorganisms present in the sample or concentrate to convert the substrate or different substrate for a sufficient time to enable detection of substrate conversion at the second pH value, and f) measuring the amount of converted substrate present in the resulting mixture, and g) based on the measured values in steps d and f determining the amount of microorganisms responsible for conversion of substrate at the first pH, the amount of microorganisms responsible for conversion of substrate at the second pH, and the total amount of microorganisms responsible for conversion of substrate.

2. The method according to claim 1, wherein the liquid sample is concentrated by passing a defined volume thereof through a filter having an influent and an effluent side and having pore sizes sufficiently small to retain the microorganisms of interest on the influent side of the filter surface3. The method according to claim 2, wherein the first composition is supplied to the influent side of the filter to flush out sample liquid from the influent side4. The method according to claim 2 or 3, wherein the second composition is added to the influent side of the filter.

5. The method according to any one of claims 2-4, wherein the amount of converted substrate is measured in a fraction of the resulting mixture that has been passed to the effluent side of the filter.

6. The method according to any one of the preceding claims, wherein the amount of any enzyme substrate present is non-limiting for the substrate conversion rate.

7. The method according to any one of the preceding claims, wherein conversion of one single substrate is measured.

8. The method according to claim 7, wherein the second composition comprises a further amount of the substrate comprised in the first composition.

9. The method according to any one of the preceding claims, wherein detection of the detectable moiety is facilitated or made possible by the change from the first pH value to the second pH value.

10. The method according to any one of the preceding claims, wherein measuring of substrate conversion comprises determination of fluorescence from a moiety released from substrate as a consequence of the enzyme activity.

11. The method according to any one of the preceding claims, wherein the first pH value is lower than the second pH value.

12. The method according to claim 11, wherein the substrate(s) is / are methylumbelliferyl derivative(s) and the moiety released is 4-methylumbelliferone.

13. The method according to claim 11 or 12, wherein the first enzyme is an acidic phosphatase and the second enzyme is an alkaline phosphatase.

14. The method according to any one of claims 1-12, wherein the substrate in the first composition is a substrate for hexosaminidase, such as p-N-acetylhexosaminidase (EC 3.2.52), and wherein the second composition comprises a substrate for phosphatase.

15. The method according to any one of the preceding claims, wherein step d) comprises that a volume of the resulting mixture is passed through the filter from the influent to the effluent side and the amount of the converted substrate is measured in said volume.

16. A kit or apparatus, which comprises i. a first composition having a first pH value and comprising a first enzyme substrate,II. a second composition having a second, higher or lower pH value than the first composition, and comprising either the first enzyme substrate or a different enzyme substrate or no enzyme substrate, ill. a filter unit, where the filter has a pore size that facilitates retention on the influent filter surface of bacteria and other microorganisms when a liquid is passed through the filter in the filter unit, and iv. means for drawing or pumping liquid sample material and the first and second compositions through the filter unit, wherein each enzyme substrate comprises a moiety, which upon enzymatic action on the substrate(s) exhibits detectable properties to allow detection of converted substrate.

17. The kit or apparatus according to claim 16, wherein the moity exhibits detectable properties which are optimal or only present at a pH value between the first and second pH values or at a pH value closer to the second pH value than to the first pH value.

18. The kit or apparatus according to claim 16 or 17, wherein the second pH value is higher than the first pH value.

19. The kit or apparatus according to claim 18, wherein each enzyme substrate is a methylumbelliferyl derivative and the moiety is 4-methylumbelliferone.

20. The kit or apparatus according to any one of claims 16-19, wherein the first and second composition comprises the same enzyme substrate.

21. The kit or apparatus according to any one of claims 16-19, wherein the first and second composition comprises substrates for different enzyme activities.

22. The kit or apparatus according to any one of claims 16-19, wherein the second composition does not comprise an enzyme substrate.

23. The kit or apparatus according to any one of claims 16-20 or 22, wherein the enzyme substrate is a phosphatase substrate24. The kit or apparatus according to claim 21, wherein the enzyme substrate in the first composition is a hexosaminidase substrate, and the substrate in the second composition is a phosphatase substrate.

25. The kit or apparatus according to any one of claims 16-24, which further comprises means for detection of the moiety / moieties, which upon enzymatic action on the substrate(s) exhibit(s) detectable properties to allow detection of converted substrate.

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