ENZYME ACTIVITY ASSAY SYSTEMS AND METHODS

Abstract

A method and a system of determining activity of a target enzyme of a sample is described. The method comprises bringing the sample in physical contact with a substrate for the target enzyme, incubating the sample with the substrate for an actual incubating time, determining a parameter value associated to enzymatic actions of the target enzyme involving the substrate, correlating the actual incubation time for the sample to at least two sets of reference data, determining a best fit standard curve correlated to the actual incubation time for the sample, and correlating the determined parameter value to the best fit standard curve and determine the target enzyme activity. Each of the at least two sets of reference data comprises data representing a standard curve for the parameter associated to the enzymatic actions of the target enzyme involving the substrate as a function of enzyme activity and correlated to an incubation time.

Claims

1. A system for determining activity of a target enzyme in a sample, the system comprises a substrate for the target enzyme, an incubator for incubating said sample with said substrate, a reader for determining a parameter associated to enzymatic actions of said target enzyme involving said substrate, and a computer system wherein the computer system is storing at least two sets of reference data, each set of reference data comprises data representing a standard curve for said parameter associated to said enzymatic actions of said target enzyme involving said substrate as a function of enzyme activity and correlated to an incubation time and an incubation time attribute representing said incubation time, wherein said at least two sets of reference data having different incubation time attribute representing different incubation time.

2. The system of claim 1, wherein the computer system is configured for receiving data representing a determined parameter for a sample and receiving data representing an associated incubation time for said sample and wherein the computer system is programmed for correlating the incubation time for said sample to said at least two sets of reference data and determining data representing a best fit standard curve correlated to said incubation time for said sample, correlating the data representing the determined parameter to the data representing the best fit standard curve and determine data representing the target enzyme activity, and transmitting said data representing the target enzyme activity to a display.

3. The system of claim 2, wherein the best fit standard curve is a calculated best fit standard curve obtained by performing a regression between two or more of said sets of reference data stored on said memory.

4. The system of any one of claims 1 to claim 3, wherein the target enzyme is a Hydrolase, a Lyase, a Ligase, a Lipase, a protease, a cellulose and/or an amylase.

5. The system of any one of claims 1 to claim 4, wherein the target enzyme is a degradative enzyme capable of degrading and/or digestion the substrate, such as glycoside hydrolase (e.g. amylase), protease, lipases and/or cellulase, amylase.

6. The system of any one of claims 1 to claim 5, wherein the substrate comprises a biopolymer such as a polymer comprising biomolecules selected from polynucleotides, polypeptides, polysaccharides or any combinations thereof, wherein the biopolymer optionally is crosslinked.

7. The system of any one of claims 1 to claim 6, wherein the substrate is a dyed substrate, a chromogenic substrate and/or a chromophore labelled substrate.

8. The system of any one of claims 1 to claim 7, wherein the parameter is an optical parameter and the reader is an optical reader, such as a spectroscope.

9. The system of any one of claims 2 to claim 8, wherein the computer system is configured for receiving at least a portion of said data representing the associated incubating time for said sample from a user via a user interface.

10. The system of any one of claims 2 to claim 9, wherein the computer system is configured for receiving at least a portion of said data representing the associated incubation time for said sample from the reader.

11. The system of any one of claims 2 to claim 10, wherein the computer system is configured for receiving said data representing the determined parameter for the sample from a user via a user interface and/or from the reader.

12. The system of any one of claims 1 to claim 11, wherein the computer system is storing at least three sets of reference data, such as 5 sets of reference data, each set of reference data comprises data representing a standard curve for said parameter associated to said enzymatic actions of said target enzyme involving said substrate correlated to an incubation time and an incubation time attribute representing said incubation time, wherein said sets of reference data having different incubation time attribute representing different incubation time.

13. The system of any one of claims 1 to claim 12, wherein each set of reference data comprises at least two data pairs of enzyme activity value versus parameter value, preferably each set of reference data comprises at least three data pairs, such as at least 5 data pairs, such as at least 8 data pairs.

14. The system of any one of claims 1 to claim 13, wherein said sets of reference data comprises at least two sets of reference data having different incubation time attribute representing incubation time that differs with at least about 30 seconds, such as at least about 1 minute, such as at least about 5 minutes, such as at least about 10 minutes.

15. The system of any one of claims 1 to claim 14, wherein the system is configured for obtaining the sample by extraction of a biological at least partly solid material, such as grain material and wherein the system further comprises an extraction container, an extraction buffer for extracting the preselected target enzyme, container, such as a filter vial with a corresponding independent filter piston or a microcentrifuge tube.

16. The system of any one of claims 1-15, wherein said at least two sets of reference data represents a standard curve associated to a selected substrate.

17. The system of any one of claims 1-16, wherein said substrate is a dyed and/or chromogenic substrate.

18. The system of any one of claims 1-17, wherein said optical reader is configured for reading at least one wavelength absorbable by a dye of said substrate.

19. A method of determining activity of a target enzyme in a sample, the method comprises bringing the sample in physical contact with a substrate for the target enzyme, incubating the sample with the substrate for an actual incubating time, determining a parameter value associated to enzymatic actions of said target enzyme involving said substrate, correlating the actual incubation time for said sample to at least two sets of reference data, determining a best fit standard curve correlated to said actual incubation time for said sample, and correlating the determined parameter value to the best fit standard curve and determine the target enzyme activity, wherein each of said at least two sets of reference data comprises data representing a standard curve for said parameter associated to said enzymatic actions of said target enzyme involving said substrate as a function of enzyme activity and correlated to an incubation time.

20. The method of claim 19, wherein the dyed and/or chromogenic substrate is specific for the target enzyme.

21. The method of claim 19 or claim 20, wherein the substrate is a gelled biopolymer substrate comprising cross-linked polymeric biomolecules selected from polynucleotides, polypeptides, polysaccharides or any combinations thereof, preferably the dyed and/or chromogenic substrate is an aerogel or a xerogel.

22. The method of any one of claims 19-21, wherein the method further comprises generating at least two sets of reference data, the method comprises generating each set of reference data by providing a plurality of reference samples with different and known enzyme activity of the target enzyme, providing a selected reference substrate for each reference sample, subjecting each of the respective reference samples to the respective selected reference substrates for a preselected reference incubation time, determining for each sample an a parameter value associated to enzymatic actions of said target enzyme involving said reference substrate, performing a regression including points of respective pairs of determined absorbance parameter and corresponding, known enzyme activity to provide a linear standard curve. wherein the preselected reference curve for generation of said respective sets of reference curves, differs from each other.

23. The method of claim 22, wherein the selected reference substrate is identical for the sample and for all reference samples.

24. The method of claim 22, wherein the plurality of material reference samples with different and known enzyme activity of the target enzyme comprises at least 3 reference samples, such as at least 5 reference samples, such as at least 8 reference samples.

25. The method of claim 22 or claim 23, wherein the selected reference substrate for the sample and for each reference sample is a dyed and/or chromogenic reference substrate.

26. The method of any one of claims 19-25, wherein the parameter value associated to enzymatic actions of said target enzyme involving said substrate is an absorbance parameter of dyed fragments released and/or generated from the substrate.

27. The method of any one of claimsclaim 22-26, wherein the actual incubation time is at least one minute, such as at least 2 minutes, such as 5 to 15 minutes.

28. The method of claim 27, wherein the preselected reference incubation time, for the generation of at least one of the sets of reference curves is from 5 minutes shorter to 5 minutes longer than the actual incubation time.

Description

BRIEF DESCRIPTION OF THE EXAMPLES AND DRAWING

[0101] The invention is being illustrated further below in connection with a few examples and embodiment and with reference to the drawings in which:

[0102] FIG. 1 shows a table of pH optimum for a number of enzymes.

[0103] FIG. 2a illustrates a system for determining activity of a target enzyme of a sample.

[0104] FIG. 2b illustrates a system for determining activity of a target enzyme of a grain material.

[0105] FIG. 3a illustrates a standard curve associated to a preselected extraction and digestion procedure.

[0106] FIG. 3b illustrates a further standard curve associated to a preselected extraction and digestion procedure.

[0107] FIG. 4a illustrates data points for five standard curves, each associated to respective preselected extraction times for extracting target enzyme from a grain material.

[0108] FIG. 4b illustrates five standard curves for five different selected extracting times.

[0109] FIG. 5a illustrates data points for six standard curves, each associated to respective preselected reference incubating times for enzymatic actions involving a substrate by respective samples containing known amount of target enzyme.

[0110] FIG. 5b show standard curves of concentration as a function of absorbance for a number of incubation times.

[0111] FIG. 6 shows determined absorbance parameter values as a function of the Ring trial WK values obtained in example 2 an example 3.

[0112] FIG. 7 shows determined absorbance parameter values as a function of the Ring trial WK values obtained in example 4.

[0113] FIG. 8 shows determined absorbance parameter values as a function of the Ring trial WK values obtained in example 5.

[0114] FIG. 9 shows determined absorbance parameter values as a function of the Ring trial WK values obtained in example 6.

[0115] FIG. 10 shows determined absorbance parameter values as a function of the DU values obtained in example 7.

[0116] FIGS. 11a and 11b shows determined absorbance parameter values as a function of the BU3/ml and BU3/g values obtained in example 8.

[0117] FIGS. 12a and 12b shows determined absorbance parameter values as a function of the BU3/ml and BU3/g values obtained in example 9.

[0118] The system illustrated in FIG. 2a for determining activity of a target enzyme of a sample, comprises a container 2 containing a substrate 1, a cuvette 3, a pipette 4, a reader 5 and a tablet 6, such as a smartphone.

[0119] In use, a sample 9 comprising a target enzyme is added into the container 2 comprising the substrate for the target enzyme. The sample is advantageously a liquid sample, e.g. obtained from a biological matter, e.g. by extraction, dissolving or diluting or without any pretreatment. The container 2 act as an incubator and the sample is incubated with the substrate for an incubating time, also referred to as an actual incubation time or the incubation time associated to this sample. The incubation time may be terminated by adding a stop agent to the container 2. After incubation, a portion of the sample is taken out from the container 2 using the pipette 4 and added to the cuvette 3. The cuvette 3 is inserted into the reader 5, which in this example is an optical reader configured for reading absorbance. The reader 5 is reading and/or determining an absorbance parameter value associated to enzymatic actions of the target enzyme involving the substrate. The absorbance value is transmitted e.g. wireless as indicated with the waves W to the tablet 6. The tablet may also be configured to transmit data to the reader 5, such as data representing the actual incubation time. The reader 5 may be calibrated e.g. using a blank sample.

[0120] A computer of the reader 5 and a computer of the tablet 6 form parts of the computer system and they may be as described above. The computer system comprises a memory storing reference data representing one or more standard curves, such as at least two sets of reference data, each set of reference data comprises data representing a standard curve for said parameter associated to said enzymatic actions of said target enzyme involving said substrate as a function of enzyme activity and correlated to an incubation time and an incubation time attribute representing said incubation time, wherein said at least two sets of reference data having different incubation time attribute representing different incubation time

[0121] The system illustrated in FIG. 2b for determining activity of a target enzyme of a grain material, comprises a milling device 17, an extracting device 18, a container 12 containing a substrate1 1, a cuvette 13, a pipette 14, a reader 15 and a tablet 16, such as a smartphone.

[0122] In use, a sample of grain material 19 comprising a target enzyme is milled in the milling device 17 e.g. as described above. The milled material or at least a portion of the milled material is moved to the extracting device 18 for extracting target enzyme into an extraction liquid e.g. as described above. At least a portion of the extraction liquid is moved to the container 12 e.g. using a method as described. The container 12 act as an incubator and the extracting liquid with extracted target enzyme is incubated with the substrate for an incubating time, also referred to as an actual incubation time or the incubation time associated to this sample. The incubation time may be terminated by adding a stop agent to the container 12. After incubation, a portion of the liquid is taken out from the container 2 using the pipette 4 and added to the cuvette 13. The cuvette 13 is inserted into the reader 15, which in this example is an optical reader configured for reading absorbance. The reader 15 is reading and/or determining an absorbance parameter value associated to enzymatic digestion of the substrate by the target enzyme. The absorbance value is transmitted e.g. wireless as indicated with the waves W to the tablet 16. The tablet may also be configured to transmit data to the reader 15, such as data representing the actual incubation time. The reader may be calibrated e.g. using a blank sample.

[0123] A computer of the reader 15 and a computer of the tablet 16 form parts of the computer system and they may be as described above. The reader 15 and the tablet 16 may be as described for the system of FIG. 2a.

[0124] FIG. 2 illustrates a system for determining activity of a target enzyme of a grain material.

[0125] The standard curve associated to a preselected extraction and digestion procedure illustrated in FIG. 3a represents beta-amylase enzyme activity in terms of diastatic power (unit Windisch-Kolbach (WK)) as a function of absorbance parameter at a preselected incubation time. The standard curve may be obtained by determining the absorbance parameter value for a number of samples with known beta-amylase enzyme activity (WK), using the preselected incubation time. Where the beta-amylase enzyme activity is determined in grain, the grain may be extracted as described above using a preselected extraction time.

[0126] The standard curve associated to a preselected extraction and digestion procedure illustrated in FIG. 3b and represent alpha amylase enzyme activity in terms of dextrinization units (DU) as a function of absorbance parameter at a preselected incubation time. The standard curve may be obtained by determining the absorbance parameter value for a number of samples with known alpha amylase enzyme activity (DU), using the preselected incubation time. Where the alpha amylase enzyme activity is determined in grain, the grain may be extracted as described above using a preselected extraction time.

[0127] The five standard curves illustrated by the data points in FIG. 4a, show standard curves for alpha amylase enzyme activity in terms of dextrinization units (DU) as a function of absorbance parameter where the standard curves are associated to respective preselected extraction times for extracting target enzyme, here alpha amylase, from a grain material.

[0128] The curves may be obtained by determining absorbance parameter values for a number of grain material samples with different and known alpha amylase enzyme activity (DU) and at different extraction times. Preferably, the incubation time used for the determinations are identical. In this example the absorbance parameter values for five grain material samples with different and known alpha amylase enzyme activity (DU) - here 20, 40, 60, 80 and 100 DU respectively were determined at extraction times of 3, 4, 5, 6 and 7 minutes.

[0129] It will be observed that the data points for each standard curve are lying on a substantially straight line.

[0130] The five standard curves illustrated in FIG. 4b for five different selected extraction times are provided using the data points of FIG. 4a. The five standard curves are each associated to respective preselected reference extracting times for extracting of alpha amylase and show absorbance parameter as a function of extraction time of extracting of alpha amylase from grain.

[0131] It can be seen that the standard curves are practically linear, which makes it relatively simple to determining a best fit standard curve for any extraction time, such as an extraction time associated to a sample under examination, where the extraction time differs from the extraction times used in generating the standard curves - e.g. an extraction time between extraction times used in generating the standard curves.

[0132] After having determined the best fit standard curve for such sample under examination, the target enzyme activity may be determined from the absorbance parameter value determined for the sample under examination.

[0133] The six standard curves illustrated by data points in FIG. 5a, is each associated to respective preselected reference incubating times for enzymatic actions involving a substrate by respective samples containing known amount in ppm of target enzyme.

[0134] The curves may be obtained by determining absorbance parameter values for a number of samples with different and known amounts of target enzyme (ppm) and at incubation times. It will be observed that the data points for each standard curve are lying on a substantially straight line.

[0135] The standard curves illustrated in FIG. 5b of concentration as a function of absorbance for a number of incubation times are obtained using the data points of FIG. 5a.

[0136] It can be seen that the six standard curves are practically linear, which makes it relatively simple to determining a best fit standard curve for any incubation time, such as an incubation time associated to a sample under examination, where the incubation time differs from the incubation times used in generating the standard curves - e.g. an incubation time between incubation times used in generating the standard curves.

[0137] After having determined the best fit standard curve for such sample under examination, the target enzyme activity may be determined from the absorbance parameter value determined for the sample under examination.

[0138] FIG. 5a. illustrates six standard curves, each associated to respective preselected reference incubating times for digestion a substrate by a fluid extract sample and representing absorbance parameter as a function of incubation time for samples with known target enzyme activity, wherein the respective preselected incubating times differs from each other with respect to preselected reference incubation time.

[0139] FIG. 5b show the concentration as a function of absorbance for a number of incubation times.

[0140] In the following examples the terms “Distatic power” and “Ring trial” has the following meaning:

[0141] Diastatic power: The malts ability to break down starches into simpler fermentable sugars during the mashing process. It is measured in Windisch-Kolbach (WK), loB or Lintner. By this definition, this is the join action of all amylases, mostly alpha amylase and beta amylase, which are the main sugar producing enzymes during mashing. However, since alpha amylase is always in excess, diastatic power is somehow correlated with beta amylase.

[0142] Ring trial: Validation of the methods of one’s lab through the collaboration of typically 10-100 different labs. It includes methods for diastatic power and alpha amylase. The main ring trials in Europe are organized by the Institute Francais de Boissons, de la Brasserie et de la Malterie (IFBM), LGC Standards, known as the Malt analytes scheme (MAPS), and the ones organized by the VLB institute in Berlin.

EXAMPLE 1

Constructing a Calibration Curve for Determining Alpha-Amylase Activity in Barley Malt Samples

[0143] The construction of calibration curves is based on grain reference samples with known reference values of enzyme activities supplied by recognized certification bodies such as EBC and IFBM.

[0144] 13 samples barley grain reference samples with known alpha-amylase activity is obtained from the European Brewery Convention and the French Institute of Beverages, Brewing and Malting. The sample is as follows:

TABLE-US-00001 Barley reference sample # known alpha-amylase activity (DU) known beta-amylase activity (WK) 1 59 258 2 55 595 3 51 250 4 48 222 5 50 390 6 40 203 7 55 267 8 64 271 9 64 269 10 66 334 11 64 331 12 24 223 13 49 269

[0145] The dextrinizing units (DU) is a standard unit in the malt industry and specified in EBC Method “4.13 α-AMYLASE CONTENT OF MALT (IM) -2006”. The diastatic power measured in Windisch-Kolbach units is specified in EBC method 4.12.1.DIASTATIC POWER OF MALT BY SPECTROPHOTOMETRY (MANUAL METHOD) - 2018.

[0146] A fluid extract reference sample is obtained from each barley malt reference sample using a preselected extraction procedure.

[0147] The preselected extraction procedure is as follows: [0148] The samples are milled by use of a lab mill, e.g. Bühler Miag Disc Mill [0149] 300 mg of the milled grain material from each barley reference sample is mixed with 50 ml liquid extraction buffer. Each sample of liquid extraction with barley reference sample is arranged in a shaker. Let the enzyme extraction go on for 5 minutes with gentle shaking at 20 rpm using an overhead shaker, for allowing the enzyme extraction for 5 minutes.

[0150] The composition of the liquid extraction buffer was a maleic acid based extraction buffer prepared according to the following protocol:

[0151] Dissolve 134.1 g of malic acid, 70 g of NaOH, and 58.4 g of NaCl in 900 mL of water (deionized or ultra-pure). Next, add 6.0 g CaCl.sub.2*H.sub.2O until complete dissolution. The pH should be adjusted to pH 5.4 by dropwise addition of concentrated (4 mol L.sup.-1) NaOH or HCl. Sodium azide (1.0 g) can be added as a preservative (antimicrobial agent). If sodium azide is added, the reagent will be stable for more than one year. If not, the shelf-life of the reagent will be 2 weeks if preserved at 4° C. This concentrated buffer should be diluted 200 times (e.g. 50 mL of concentrated solution for a final volume of 1000 mL) in order to be used in the extraction protocol.

[0152] The extraction was performed at room temperature (21° C.).

[0153] Thereafter a fluid extract reference sample of 250 microliter is taken of each liquid extraction buffer with extracted enzyme using a pipette suitable for the purpose. Each sample was carefully aspirated from the liquid phase and thereby minimizing the risk of introducing solid matter in the pipette tip. After proper aspiration, the sample was dispensed in the provided filter vial containing the substrate and further diluted by adding 250 microliter of the above mentioned buffer.

[0154] The substrates were produced by first dyeing polysaccharides with one of the four chlorotriazine dyes (red, blue, green or yellow) via nucleophilic aromatic substitution. The polysaccharides were then cross-linked with 1,4-butanediol diglycidyl ether via base-catalysed epoxide opening. The resulting materials are hydrogels, which can be easily dispensed using syringes into 96-well filter plates or vials. Additional info about production of such substrate may be found in Kračun, S.K., et al., A new generation of versatile chromogenic substrates for high-throughput analysis of biomass-degrading enzymes. Biotechnology for Biofuels, 2015. 8(1): p. 70.

[0155] The alpha-amylase in the sample will digest the substrate and develop a blue colored solution with an intensity related to the enzymatic activity as a function of incubation time.

[0156] The incubation was performed at room temperature applying a reference incubating time of 5 minutes with gentle shaking at 20 rpm using an overhead shaker.

[0157] For each sample, the reference incubation time is terminated by suction of the fluid extract reference sample with released substrate fragments and dye over the filter in each container and collecting the fluid extract reference sample in a cuvette.

[0158] A further cuvette is supplied by a “blind” sample of the liquid extraction buffer without any extracted enzyme. Instead of a blind sample of liquid extraction buffer, a blind sample of pure water could have been used.

Measurement

[0159] Each reference sample including the blind is subjected to a spectrometer to determine the intensity of transmitted light of a light source comprising a wavelength absorbable by the dye.

[0160] An absorbance parameter is determined for each reference samples by withdrawing the light intensity transmitted through the blind from the light intensity transmitted through the respective samples.

[0161] Thereafter the respective absorbance parameter is plotted as a function of alpha amylase activity (DU).

TABLE-US-00002 Barley reference sample # known alpha-amylase activity (DU) absorbance (# transmitted light intensity - blind transmitted light intensity) 1 59 1.22 2 55 1.2 3 51 1.16 4 48 1.1 5 50 1.14 6 40 1.0

[0162] The absorbance is determined according to the formula:

[00001]$A={\log }_{10}\frac{{\text{Φ}}_{\text{e}}^{\text{i}}}{{\text{Φ}}_{\text{e}}^{\text{t}}}=-{\log }_{10}T,$

where

[00002]${\text{Φ}}_{\text{e}}^{\text{t}}$

is the radiant flux transmitted by that material,

[00003]${\text{Φ}}_{\text{e}}^{\text{i}}$

is the radiant flux received by that material,

[00004]$T={\text{Φ}}_{\text{e}}^{\text{t}}/{\text{Φ}}_{\text{e}}^{\text{i}}$

is the transmittance of that material.

Example 2

Ring Trial Validation - Extraction Time

[0163] Ring trial validations using five different milled malt samples having different WK values were performed.

[0164] The five samples of milled barley malt with different and known WK value were as follows:

TABLE-US-00003 Sample Sample 1 Sample 2 Sample 3 Sample 4 Sample 5 WK 203 267 334 379 60

[0165] The samples were tested according to the following protocol: [0166] Weigh 200 mg of milled malt. [0167] Extraction with extraction buffer for 10 minutes at 35° C. [0168] Syringe filtration with 0.45 .Math.m filter. Collect the filtrate. [0169] Incubation of 250 .Math.L filtrate with substrate for an incubation time at 35° C. [0170] Stop the enzymatic cleaving by adding a stopping reagent [0171] Read the resulting liquid at 410 nm in a spectrophotometer.

[0172] The extraction buffer used was the maleic acid based extraction buffer described in example 1. For each sample, 25 mL of extraction buffer was used together with 35 mg Dithiothreitol.

[0173] The substrate used was a glucose-based oligosaccharide conjugated with a chromophore, able to absorb visible light at 410 nm when the pH is higher than 9, mixed with a glucosidase. By the action of the enzyme of interest, in this case beta amylase, the oligosaccharide is then accessible to the glucosidase, which then makes the chromophore free of glucose molecules. Such chromophore would then change color by the addition of the stopping reagent, and its intensity will depend directly on how much the enzyme of interest cleaves the oligosaccharide.

[0174] The incubation time used was 7 minutes.

[0175] The stopping agent used was an alkaline sodium carbonate solution.

[0176] The spectrophotometer used was the spectrometer sold by Glycospot under the trade name SIRIUS™ .

[0177] The absorbance parameter for each sample was determined by withdrawing the light intensity transmitted through a blind from the light intensity transmitted through the respective samples.

[0178] Each test was performed in triplicate and the average absorbance parameter values were determined.

[0179] Thereafter the respective absorbance parameter values were plotted as a function of the Ring trial WK values. The plot is shown in FIG. 6.

[0180] The result indicates a good correlation with the ring trial samples even with a relatively low extraction time, here 10 minutes. It is assessed that an even lower extraction time could have been applied and still maintaining a good correlation with the ring trial.

Example 3

Reference Curve for Barley Malt - Diastatic Power Range 30-500 WK

[0181] The test results obtained in example 2 and shown in FIG. 8 were applied as reference curve for barley malt.

[0182] The tests are repeated using different incubation times including the incubation times 3 minutes, 10 minutes and 20 minutes to thereby prepare additional sets of reference data correlated to respective incubation times as described above.

Example 4

Ring Trial Validation - Incubation Time

[0183] Ring trial validations using three different milled malt samples having different WK values were performed.

[0184] The samples were tested according to the protocol given in example 2, where the buffer, the substrate, the stopping agent and the reading out were as in example 2.

[0185] Each sample was tested with three different incubation times, namely 3 minutes, 5 minutes and 7 minutes respectively.

[0186] Each test was performed in triplicate and the average absorbance parameter values were determined.

[0187] Thereafter the respective absorbance parameter values were plotted as a function of the Ring trial WK values. The plot is shown in FIG. 7.

[0188] The results show a very good correlation with the ring trial samples for each of the three incubation times

[0189] Thus, by applying data pairs of the WK-values/ determined absorbance parameter values as reference data, the computer system may be programmed to determine the WK value for an unknown sample tested according to the protocol and with an incubation time which may be any time within a range 3-7 minutes and at least some minutes beyond the 3-7 minutes incubation time.

Example 5

Preparing Reference Curve for Barley Malt - Diastatic Power Range 500-100 WK

[0190] Three samples of milled barley malt with different and known WK value were tested:

TABLE-US-00004 Sample Sample 1 Sample 2 Sample 3 WK 893* 608 746

[0191] The samples were tested according to the protocol given in example 2, where the buffer, the substrate, the incubation time, the stopping agent and the reading out were as in example 2.

[0192] *The actual WK value of the sample 1 material was 595, however, for test of this sample 50% more barley malt (i.e. 300 mg) was used to represent the 895 WK value.

[0193] Each test was performed in triplicate and the average absorbance parameter values were determined.

[0194] Thereafter the respective absorbance parameter values were plotted as a function of the Ring trial WK values. The plot is shown in FIG. 8.

[0195] The tests may be repeated using different incubation times. For providing two or more sets of reference data correlated to respective incubation times as described above.

Example 6

Preparing Reference Curve for Wheat Malt - Diastatic Power Range 200-500 WK

[0196] Four samples of milled wheat malt with different and known WK value were tested:

TABLE-US-00005 Sample Sample 1 Sample 2 Sample 3 Sample 4 WK 223 269 390 445

[0197] The samples were tested according to the protocol given in example 2, where the substrate, the incubation time, the stopping agent and the reading out were as in example 2.

[0198] The extraction buffer was as in example 2 but the amount applied was 50 mL together with 70 mg Dithiothreitol/sample.

[0199] Each test was performed in triplicate and the average absorbance parameter values were determined.

[0200] Thereafter the respective absorbance parameter values were plotted as a function of the Ring trial WK values. The plot is shown in FIG. 9.

[0201] The tests may be repeated using different incubation times. For providing two or more sets of reference data correlated to respective incubation times as described above

Example 7

Preparing Reference Curve for Alpha Amylase Activity for Barley Malt (10-100 DU)

[0202] Three different samples of barley malt with known DU values were subjected to an extraction according to the following extraction protocol: [0203] Weigh 200 mg of milled malt. [0204] Extraction with extraction buffer for 10 minutes at 35° C. [0205] Syringe filtration with 0.45 .Math.m filter. Collect the filtrate.

[0206] The three samples were subjected to a dilution series to provide in total 9 diluted samples, three from each of the samples A, B and C as listed below:

TABLE-US-00006 Sample mL buffer 3 —dilution step Dilution ratio (always pipette 250 .Math.L of the extraction) Known and calculated-DU Sample A 3 - 51 DU 45 181 10,4 9 37 51 4,5 19 99,3 Sample B 6-40 DU 36 145 10,2 12 49 30,2 9 37 40,0 Sample C 10 - 65,6 DU 27 109 22,3 9 37 65,6 7 29 83,7

[0207] The samples were tested according to the following protocol: [0208] Incubation of 250 .Math.L filtrate with substrate for an incubation time at 35° C. [0209] Stop the enzymatic cleaving by adding a stopping reagent [0210] Read the resulting liquid at 410 nm in a spectrophotometer.

[0211] The buffer used for the dilution series was the maleic acid based extraction buffer described in example 1.

[0212] The substrate used was a glucose-based oligosaccharide conjugated with a chromophore, able to absorb visible light at 410 nm when the pH is higher than 9, mixed with a glucosidase. By the action of the enzyme of interest, in this case alpha amylase, the oligosaccharide is then accessible to the glucosidase, which then makes the chromophore free of glucose molecules. Such chromophore would then change color by the addition of the stopping reagent, and its intensity will depend directly on how much the enzyme of interest cleaves the oligosaccharide.

[0213] The incubation time used was 7 minutes.

[0214] The stopping agent used was an alkaline sodium carbonate solution.

[0215] The spectrophotometer used was the spectrometer sold by Glycospot under the trade name SIRIUS™ .

[0216] The absorbance parameter for each sample was determined by withdrawing the light intensity transmitted through a blind from the light intensity transmitted through the respective samples.

[0217] Each test was performed in triplicate and the average absorbance parameter values were determined.

[0218] Thereafter the respective absorbance parameter values were plotted as a function of the DU values. The plot is shown in FIG. 10.

[0219] The tests may be repeated using different incubation times. For providing two or more sets of reference data correlated to respective incubation times as described above.

Example 8

Preparing Reference Curve for Beta Amylase Activity in Malt in Betamyl-3 Units BU3

[0220] Betamyl-3 unit is the typical unit for beta amylase activity.

[0221] A single sample of barley malt with known BU3 value was subjected to an extraction according to the following extraction protocol: [0222] Weigh 200 mg of milled barley malt. [0223] Extraction with extraction buffer for 10 minutes at 35° C. [0224] Syringe filtration with 0.45 .Math.m filter. Collect the filtrate.

[0225] The sample were subjected to a dilution series to provide in total 6 diluted samples.

[0226] Each of the 6 dilution samples was tested according to the following protocol: [0227] Incubation of 250 .Math.L filtrate with substrate for an incubation time at 35° C. [0228] Stop the enzymatic cleaving by adding a stopping reagent [0229] Read the resulting liquid at 410 nm in a spectrophotometer.

[0230] The buffer used for the dilution series was the maleic acid based extraction buffer described in example 1.

[0231] The substrate used was a glucose-based oligosaccharide conjugated with a chromophore, able to absorb visible light at 410 nm when the pH is higher than 9, mixed with a glucosidase. By the action of the enzyme of interest, in this case beta amylase, the oligosaccharide is then accessible to the glucosidase, which then makes the chromophore free of glucose molecules. Such chromophore would then change color by the addition of the stopping reagent, and its intensity will depend directly on how much the enzyme of interest cleaves the oligosaccharide.

[0232] The incubation time used was 7 minutes.

[0233] The stopping agent used was an alkaline sodium carbonate solution.

[0234] The spectrophotometer used was the spectrometer sold by Glycospot under the trade name SIRIUS™ .

[0235] The absorbance parameter for each sample was determined by withdrawing the light intensity transmitted through a blind from the light intensity transmitted through the respective samples.

[0236] Each test was performed in triplicate and the average absorbance parameter values were determined.

[0237] Thereafter the respective absorbance parameter values were plotted as a function of the BU3 values. The plot is shown in FIGS. 11a and 11b. FIG. 11a shows the respective absorbance parameter values versus the BU3/mL value and FIG. 11b shows the respective absorbance parameter values versus the BU3/g value.

[0238] The tests may be repeated using different incubation times. For providing two or more sets of reference data correlated to respective incubation times as described above.

Example 9

Preparing Reference Curve for Beta Amylase Activity in Malt Extract

[0239] The test of example 8 was repeated but with the difference that on 1 g of milled barley malt was extracted using 25 mL extraction buffer and wherein the dilution series was providing 4 diluted samples.

[0240] The respective absorbance parameter values were plotted as a function of the BU3 values. The plot is shown in FIGS. 12a and 12b. FIG. 12a shows the respective absorbance parameter values versus the BU3/mL value and FIG. 12b shows the respective absorbance parameter values versus the BU3/g value.

[0241] The tests may be repeated using different incubation times. For providing two or more sets of reference data correlated to respective incubation times as described above.