METHOD OF DETERMINING AMYLASE CONCENTRATIONS

Abstract: A method for determining amylase concentrations is provided, comprising the steps of: (a) incubating a part of a specimen having an unknown amylase concentration at a specified temperature in a water-miscible buffered starch solution containing starch at a concentration of between 20-200 mg/litre; (b) allowing the thus-obtained mixture to react for a specified time period; (c) adding a portion of an aqueous iodine solution to this mixture to obtain a first sample; (d) preparing a second sample by adding another part of the specimen to a mixture comprising a second portion of the iodine solution and a second portion of the starch solution; (e) measuring the optical density of both samples at a wavelength between 300-800 nm; and(f) calculating the amylase activity in the specimen using a calibration curve,wherein both the first and the second sample containequal concentrations of the specimen and the starch solution. The time needed for carrying out this method is less than 10 minutes, preferably less than 6 minutes. This method is suitable for monitoring the amylase concentration in industrial cleaning processes.

Description: Method of determining amylase concentrations Field of the invention The present invention relates to a method of determining amylase concentrations, especially the amylase concentrations of amylase-containing wash liquor applied in a machine warewashing process. More in particular, the invention relates to a method of determining amylase concentrations, wherein a starch solution and an iodine solution are applied. Background of the invention For a long time, enzymes, particularly amylase enzymes, have been known as effective ingredients of mechanical warewashing compositions for domestic use. More recently, amylase enzymes have also been disclosed as components in mechanical warewashing compositions, for use in industrial multi-tank or single-tank machines. For instance, WO-96/16152 discloses a cleaning system for a multi-tank warewashing machine, wherein amylase enzyme and bleach are dosed into different wash tanks or zones of the machine. Furthermore, WO-99/34723 discloses a cleaning system for an industrial warewashing machine, having a bleach component and an enzyme component, wherein the enzyme component is introduced into a wash zone or step and the bleach component is introduced into a post-wash rinse zone or step. When commercially using such industrial mechanical warewashing processes, it is important that the use concentration of the amylase enzyme can be determined regularly using a quick, simple and reliable technique. In US Patent 3,888,739 such a technique is disclosed, particularly for use in hospital laboratories. The method described in this document utilizes starch and iodine solutions and is comprised of the following steps. A starch solution is prepared from a freeze-dried starch substrate. A specimen having an unknown amylase concentration is incubated at a specified temperature in this starch solution. After a specified time, an iodine solution is admixed with the starch solution containing the specimen to stop the reaction between the enzyme and the starch. Another portion of the iodine solution is admixed into another portion of the starch solution without the presence of any specimen therein. The optical density of the starch-iodine solution containing the specimen is compared against the optical density of the starch-iodine solution without the specimen. The difference in optical densities is used as a relative measure of how much amylose (present in the starch) has reacted with the amylase, which is a function of the amylase concentration of the specimen. The entire method of US-A-3, 888 , 739, as described above, starting from the time the freeze-dried starch is reconstituted until the amylase concentration is determined, is performed in about 15 minutes. It is an object of the present invention to further shorten the time period for amylase activity determination. It is another object of the invention to find a method for amylase activity determination which is suitable for monitoring the amylase concentration in industrial cleaning processes such as the process carried out in a multi-tank or single-tank warewashing machine, whereby interferences caused by the presence of soil and enzyme stabilizing species, such as sulphite, are avoided. It has been surprisingly found that these objects can be achieved by applying the method of claim 1. Definition of the invention Accordingly, the present invention provides a method for determining amylase concentrations, comprising the steps of: (a) incubating a part of a specimen having an unknown amylase concentration at a specified temperature in a water-miscible buffered starch solution containing starch at a concentration of between 20 -200 mg/litre; (b) allowing the thus-obtained mixture to react for a specified time period; (c) adding a portion of an aqueous iodine solution to this mixture to obtain a first sample; (d) preparing a second sample by adding another part of the specimen to a mixture comprising a second portion of the iodine solution and a second portion of the starch solution; (e) measuring the optical density or remission of both samples at a wavelength between 300-800 nm; and (f) calculating the amylase activity in the specimen using a calibration curve, wherein both the first and the second sample contain equal concentrations of the specimen and the starch solution, and wherein the time needed for carrying out the method is less than 10 minutes. Detailed description of the invention It is highly desirable that the method of the invention can be carried out in a short time period. Reason is that said method has been developed for use in industrial cleaning processes, particularly those applied in multi-tank or single-tank warewashing machines, wherein amylase enzymes are used. In those processes, it is desirable to be able to immediately adjust the enzyme dosage on the basis of the outcome of the amylase activity determination. The present invention provides a method for accurately and simply determining the amylase activity within a sufficiently short time period for enabling a proper and timely adjustment of the enzyme dosage if needed. The method of the invention is carried out within a time period of 10 minutes. Preferably, the duration of said method is less than 8 minutes, more preferably less than 6 minutes, a time period of less than 4 minutes being most preferred. In the method of the invention, two types of sample are applied: a first type of sample being the 'test' sample, in which a part of specimen is allowed to react with a first portion of buffered starch solution for a specified time period before an aqueous iodine solution is added, and a second type of sample which is used as a reference sample. Said second type of sample is prepared by adding the rest of the specimen to a mixture of iodine solution and buffered starch solution, whereby the presence of the iodine solution prevents any reaction to take place between the specimen and the starch solution. It can be seen that both the 'test' sample and the reference sample contain equal concentrations of buffered starch solution and specimen. The presence of equal amounts of specimen in both types of sample is needed in order to be able to correct for any interference of the optical density measurement caused by the presence of soil and enzyme stabilizing species such as sulfite and other reducing materials. The buffered starch solution used in the method of the invention is a starch solution containing a suitable buffer and having a pH in the range of 5-11, preferably 6-8, a pH of around 7 being most preferred. An effective buffer for said buffered starch solution is a phosphate buffer, preferably containing a mixture of disodiu hydrogen orthophosphate and monosodium dihydrogen orthophosphate . Said buffered starch solution is preferably an aqueous solution. Furthermore, for reasons of reproducibility of the method of the invention the starch solution is desirably an isotropic aqueous solution. Preferably, the buffered starch solution is an aqueous solution containing starch at a concentration of 40-100 mg/liter. This starch is preferably readily soluble in water at room temperature, and will normally produce a definite blue color upon the addition of an aqueous iodine solution thereto. The method of the invention, in particular steps (a) and (b) thereof, can be suitably carried out at a temperature between about 10°C and 60°C, preferably at a temperature of at most 30°C, more preferably at room temperature. The reaction of step (b) of the method of the invention between the specimen and the buffered starch solution is preferably carried out for a time period of 1-5 minutes, more preferably 2-4 minutes, a time period of about 3 minutes being most preferred. This reaction time period can be adjusted by selecting the moment of starting step (c) by adding the aqueous iodine solution, since addition of said iodine solution will stop said reaction immediately. In step (e) of the method of the invention, the optical density or remission of both types of samples (expressed as % transmission or % remission) is measured at a wavelength of 300-800 ran, preferably 500-650 nm. For practical reasons, the light remission is preferably measured using a portable reflectometer . Subsequently, the delta optical density or remission being the difference between the optical density or remission values obtained with the test sample respectively the reference sample, is calculated. In step (f) , the amylase activity of the specimen concerned is calculated by interpolation using the thus-obtained optical density or remission and a calibration curve showing the relationship between delta optical density and amylase activity (for instance, expressed in Maltose Units (MU) /liter) . In a preferred embodiment of the invention, the buffered starch solution is prepared by dissolving 50 mg/liter of starch (ex Merck KGaA, Darmstadt, art. nr.1252) in 200 mM phosphate buffer at room temperature and a pH of 6.8. This phosphate buffer consists of 100 mM disodium hydrogen phosphate (Na 2 HP0 4 .7H 2 0) and 100 mM sodium dihydrogen phosphate (Na H 2 P0 4 ) . The aqueous iodine solution for use in this preferred embodiment is prepared by dissolving 1.2 mg/ml of a complex of iodine and polyvinyl pyrrolidone (PVP) (ex Aldrich) in 400 mM glycine and 20% vol. ethanol at a pH of 1.5. Said PVP, glycine and ethanol assist in stabilising and solubilising the iodine. The calibration curve used in this preferred embodiment is prepared as follows. Standard enzyme activity solutions having alpha-amylase concentrations ranging from 0-60 ppm Termamyl 300 LDX (ex NOVO) , are prepared and the activity thereof is checked using a pocket reflectometer (RQflex® ex Merck KGaA, art. nr. 16970) or an enzyme analyzer, e.g. Cobas Mira, ex Roche Diagnostics. 100 μl samples of each of these standard enzyme solutions are added to test tubes filled with a buffered starch solution as prepared above, having a temperature of 20 °C. Immediately after the addition of the standard solutions the timer is started. After exactly 3 minutes, 1 ml of iodine solution, as prepared above, is added. Thus standard test samples having varying alpha-amylase activities are obtained. In addition, 'blank' samples are prepared by adding to new test tubes 1 ml of iodine solution and lOOμl of each of the standard enzyme solutions. The remission or optical density of all thus-prepared samples is measured at a wavelength of 576nm and is plotted (see Figure 1). Subsequently, the delta optical densities for each of the standard enzyme solutions -being the difference between the optical densities obtained with the standard test sample concerned and the 'blank' sample concerned- is calculated. Thereafter, the delta optical densities so obtained are plotted as a function of the alpha-amylase activity of the standard enzyme solutions, so as to obtain a calibration curve for alpha amylase. Via linear regression, a best fit can be calculated for said calibration curve. This calibration curve can be used in the method of the invention, when applying a reaction time of 3 minutes and a temperature of 20° C in step (b) of said method. In this preferred embodiment, the actual activity determination of alpha-amylase present in the wash liquor of a warewashing machine, is performed as follows. A sample is taken from this wash liquor. A test tube containing a buffered starch solution as prepared above and having a temperature of 20 °C is taken and 100 μl of this wash liquor sample is added to the starch solution. Immediately, the timer is started. The test tube is swirled gently. After exactly 3 minutes, 1 ml of iodine solution as prepared above is added to the contents of the test tube, so as to obtain the "test" sample. Another test tube containing the same starch solution and 1 ml of the same iodine solution is taken, and lOOμl of the wash liquor sample is added so as to obtain the 'reference' sample. The optical densities of both the 'test' sample and the 'reference' sample are measured and the delta optical density being the difference between the thus-obtained values is calculated. The activity of the alpha-amylase present in the wash liquor sample is determined by interpolation using this delta optical density and the above-prepared calibration curve .

Claims: Claims 1. A method for determining amylase concentrations, comprising the steps of: (a) incubating a part of a specimen having an unknown amylase concentration at a specified temperature in a water-miscible buffered starch solution containing starch at a concentration of between 20-200 mg/litre; (b) allowing the thus-obtained mixture to react for a specified time period; (c) adding a portion of an aqueous iodine solution to this mixture to obtain a first sample; (d) preparing a second sample by adding another part of the specimen to a mixture comprising a second portion of the iodine solution and a second portion of the starch solution; (e) measuring the optical density of both samples at a wavelength between 300-800 nm; and (f) calculating the amylase activity in the specimen using a calibration curve, wherein both the first and the second sample contain equal concentrations of the specimen and the starch solution, and wherein the time needed for carrying out the method is less than 10 minutes. 2. A method according to claim 1, wherein the time needed for carrying out said method is less than 6 minutes. 3. A method according to claim 1 or 2, wherein the buffered starch solution has a pH in the range of 6-8. 4. A method according to any of claims 1-3, wherein the buffered starch solution is an aqueous solution containing starch at a concentration of between 40-100 mg/liter. 5. A method according to any of claims 1-4, wherein said method is carried out at room temperature. 6. A method according to any of claims 1-5, wherein the reaction of step (b) between the specimen and the buffered starch solution is carried out for a time period of 2-4 minutes . 7. A method according to claim 1 or 2, wherein ' the optical density is measured using a portable reflectometer . 8. A method according to any of claims 1-6, used for determining the amylase concentration of a wash liquor applied in an industrial machine warewashing process. 9. A method accoirding to claim 8, used for determining the amylase concentration in the presence of soil and enzyme stabilizing species, such as sulphite.

Inventors: DOHMEN ROGER, LINXWEILER WINFRIED, DOHMEN, ROGER, LINXWEILER, WINFRIED
Applicants: LEVER HINDUSTAN LTD, UNILEVER NV, UNILEVER PLC, HINDUSTAN LEVER LTD., UNILEVER N.V.
Application year: 2001
Main classification: C12Q1
IPC codes: C12Q1, G01N21