SYSTEM AND METHOD FOR DETERMINING ENZYME ACTIVITY IN GRAIN MATERIAL

Abstract

A method of determining activity of a target enzyme of a grain material is described. The method comprises providing a fluid extract sample of the grain material using a preselected extraction procedure, providing a dyed and/or chromogenic substrate for the target enzyme, subjecting the fluid extract sample to the substrate for a preselected incubating time, and determining the target enzyme activity of the grain material. The extraction time is relatively short while still obtaining high accuracy.

Claims

1. A method of determining activity of a target enzyme of a grain material, the method comprises providing a fluid extract sample of the grain material using a preselected extraction procedure, providing a dyed and/or chromogenic substrate for the target enzyme, subjecting the fluid extract sample to the substrate for a preselected incubating time, and determining the target enzyme activity of the grain material, wherein the preselected extraction procedure comprises milling the grain material to a predetermined particle size distribution, preparing an extract from the milled substance by a method comprising thoroughly mixing a preselected amount of the milled grain material with a preselected amount of liquid extraction buffer and allowing the enzyme extraction for a preselected extraction time of up to about 20 minutes, and taking the fluid extract sample of the liquid extraction buffer, and wherein the step of determining the enzyme activity of the grain material comprises determining an absorbance parameter of dye released from the substrate into the fluid extract sample and/or colored reaction product formed, correlating the determined absorbance parameter to a standard curve associated to the preselected extraction procedure and representing absorbance parameter as a function of target enzyme activity.

2. The method of claim 1, wherein the step of milling the grain material comprises milling the grain material to a particle size where at least 90% by weight, such as at least 95% of the grain material passes a sieve of 0.5 mm (mesh 35) or less, such as, a sieve of 0.4 mm (mesh 40) or less, such as, a sieve of 0.3 mm (mesh 50) or less, such as, a sieve of 0.2 mm (mesh 70) or less.

3. The method of claim 1 or claim 2, wherein the step of milling the grain material comprises milling the grain material using a mill, such as a disc mill, a grist mill, a coffee mill and/or a Hammer mill.

4. The method of any one of the preceding claims, wherein the milling comprises milling a preselected amount of grain material for a preselected time.

5. The method of any one of the preceding claims, wherein the thoroughly mixing of a preselected amount of the milled grain material with a preselected amount of liquid extraction buffer comprises withdrawing at least about 10 mg, such as from about 0.1 g to about 5 g, such as from about 0.3 g to about 1 g of the milled grain material and mixing it with from about 5 ml to about 0.5 L per gram milled grain material, such as from about 10 ml to about 0.2 L per gram milled grain material.

6. The method of any one of the preceding claims, wherein the liquid extraction buffer is an aqueous buffer having a pH value of from about 4 to about 10, such as from about 5 to about 8.

7. The method of any one of the preceding claims, wherein the liquid extraction buffer comprises a reducing agent, such as Dithiothreitol (DTT) and/or tris(2-carboxyethyl)phosphine (TCEP).

8. The method of any one of the preceding claims, wherein the thoroughly mixing of a preselected amount of the milled grain material with a preselected amount of liquid extraction buffer comprises, shaking or stirring the mixture for a part of or for the entire extraction time.

9. The method of any one of the preceding claims, wherein the preselected extraction time is up to about 15 minutes, such as up to about 10 minutes, such as up to about 8 minutes, such as from about 2 to about 6 minutes.

10. The method of any one of the preceding claims, wherein the extraction is performed at a temperature from about 10 to about 40° C., such as from about 10 to about 30° C., such as from about 18 to about 24° C.

11. The method of any one of the preceding claims, wherein the preselected extraction time is terminated by taking the fluid extract sample of the liquid extraction buffer, preferably the fluid extract sample is essentially free of solids, such as free of particulate grain material.

12. The method of any one of the preceding claims, wherein the fluid extract sample is added to the substrate within 10 minutes from taking the fluid extract sample of the liquid extraction buffer, preferably within 5, such as within 2 minutes, such as immediately after taking the fluid extract sample of the liquid extraction buffer.

13. The method of any one of the preceding claims, wherein the preselected incubation time is up to about 30 minutes, such as from about 1 minute to about 20 minutes, the incubation time is preferably terminated by removing the non-digested substrate from the sample and/or by adding a stop agent.

14. The method of any one of the preceding claims, wherein the method comprises filtering of solid parts of the substrate prior to determining the absorbance parameter of dye released from the substrate into the fluid extract sample and/or colored reaction product formed, preferably the incubation time is terminated by filtering of solid parts of the substrate.

15. The method of any one of the preceding claims, wherein the determining of the target enzyme activity of the grain material comprises measuring the absorbance parameter of the fluid extract sample with released dye, wherein the absorbance parameter comprises at least one wavelength absorbable by the dye.

16. The method of any one of the preceding claims, wherein the standard curve further is associated to the selected dyed and/or chromogenic substrate and preselected incubation time.

17. The method of any one of the preceding claims, wherein the target enzyme is α-Amylase, β-Amylase, Limit Dextrinase, Beta-glucanase, lipase, cellulase, xylanase, protease or any combinations comprising one or more of these.

18. The method of any one of the preceding claims, wherein the grain material is selected from one or more of cereals, nuts, legumes, spices and any combinations comprising at least one of these.

19. The method of any one of the preceding claims, wherein the grain material is barley, wheat, rye or oat or any combinations comprising one or more of these.

20. The method of any one of the preceding claims, wherein the dyed and/or chromogenic substrate is specific for the target enzyme.

21. The method of any one of the preceding claims, wherein the selected dyed and/or chromogenic 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 the preceding claims, wherein the method further comprises generating the standard curve, wherein the generation of the standard curve comprises providing a plurality of grain material reference samples with different and known enzyme activity of the target enzyme, providing a fluid extract reference sample of each of the respective grain material reference samples using a preselected reference extraction procedure, providing a selected dyed and/or chromogenic reference substrate for the target enzyme for each of the fluid extract reference sample, subjecting each of the respective fluid extract reference samples to the respective selected dyed and/or chromogenic reference substrates for a preselected reference incubation time, determining an absorbance parameter of dye released from each of the respective reference substrates into the fluid extract sample and/or colored reaction product formed, performing a regression including points of respective pairs of determined absorbance parameter and corresponding, known enzyme activity to provide a linear standard curve.

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

24. The method of claim 22 or claim 23, wherein the selected dyed and/or chromogenic reference substrate are identical to the selected dyed and/or chromogenic substrate.

25. The method of claim 22 or claim 23, wherein the preselected incubation time for the fluid extract reference samples are from 5 minutes shorter to 5 minutes longer than the incubation time for the fluid extract samples, such as from 1 minute shorter to 1 minute longer, such as from 0.5 minutes shorter to 0.5 minutes longer.

26. A system for determining activity of a target enzyme of a grain material, the system 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, a selected dyed and/or chromogenic substrate for said preselected target enzyme, a spectroscope comprising a light source and an optical reader and a computer system, wherein the computer system comprises a memory storing data representing a standard curve associated to a preselected reference extraction procedure for extracting target enzyme from a grain material and representing absorbance parameter as a function of target enzyme activity, wherein the preselected reference extraction procedure comprises milling the grain material to a predetermined particle size determine by sieving, preparing an extract from the milled substance by a method comprising thoroughly mixing a preselected amount of the milled grain material with a preselected amount of liquid extraction buffer and allowing the enzyme extraction for a preselected reference extraction time up to about 20 minutes, and taking the fluid extract sample of the liquid extraction buffer.

27. The system of claim 26, wherein the standard curve further is associated to the selected dyed and/or chromogenic substrate and preselected incubation time.

28. The system of claim 26 or claim 27, wherein said spectroscope is arranged for determining an absorbance parameter of a sample in a sample container in reflecting mode and/or in transmitting mode.

29. The system of any one of claims 26-28, wherein said light source of said spectroscope is configured for emitting at least one wavelength absorbable by said dye.

30. The system of any one of claims 26-29, wherein said optical reader is configured for reading at least one wavelength absorbable by said dye.

31. The system of any one of claims 26-30, wherein said computer is configured for receiving data from the spectroscope representing a determined absorbance parameter, correlating the data representing the determined absorbance parameter to the data representing the standard curve and determine data representing the target enzyme activity, and transmitting said data representing the target enzyme activity.

32. The system of any one of claims 26-31, wherein said computer is programmed for timely organizing an assay for determining of target enzyme activity comprising receiving data representing a start incubation time, transmitting data representing a stop incubation time after the preselected incubation time of receiving the start incubation time, receiving data from the spectroscope representing a determined absorbance parameter, correlating the data representing the determined absorbance parameter to the data representing the standard curve and determine data representing the target enzyme activity, and transmitting said data representing the target enzyme activity.

33. The system of any one of claims 26-31, wherein said computer is programmed for timely organizing an assay for determining of target enzyme activity comprising receiving data representing a start extraction time, transmitting data representing a stop extraction time after the preselected extraction time of receiving the start extraction time, receiving data representing a start incubation time, transmitting data representing a stop incubation time after the preselected incubation time of receiving the start incubation time, receiving data from the optical reader representing a determined absorbance parameter, correlating the data representing the determined absorbance parameter to the data representing the standard curve and determine data representing the target enzyme activity, and transmitting said data representing the target enzyme activity.

34. The system of any one of claims 26-33, wherein said computer is programmed for timely organizing an assay according to the method of any one of claims 1-25.

35. The system of any one of claims 26-34, wherein said memory of the computer stores data representing two or more standard curves, each associated to respective preselected extraction procedures for extracting target enzyme from a grain material and representing absorbance parameter as a function of target enzyme activity, wherein the respective preselected extraction procedures for extracting target enzyme from a grain material differs from each other with respect to preselected reference extraction time.

36. The system of claim 35, wherein the computer is configured for receiving data representing a start extraction time, receiving data representing a stop extraction time, determining the extraction time, correlating the extraction time to the two or more standard curves and determining a best fit standard curve, correlating the data representing the determined absorbance 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.

37. The system of claim 36, wherein the best fit standard curve is a calculated best fit standard curve obtained by performing a regression between two or more of said standard curves stored on said memory.

38. The system of any one of claims 26-37, wherein said memory of the computer stores data representing two or more standard curves, each associated to respective preselected reference incubation procedures for digestion a substrate by a fluid extract sample and representing absorbance parameter as a function of target enzyme activity, wherein the respective preselected reference incubation procedures for digestion a substrate by a fluid extract sample differs from each other with respect to preselected reference incubation time.

39. The system of claim 38, wherein the computer is configured for receiving data representing a start incubation time, receiving data representing a stop incubation time, determining the incubation time, correlating the incubation time to the two or more standard curves and determining a best fit standard curve, correlating the data representing the determined absorbance 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.

40. The system of claim 39, wherein the best fit standard curve is a calculated best fit standard curve obtained by performing a regression between two or more of said standard curves stored on said memory.

Description

BRIEF DESCRIPTION OF THE EXAMPLES AND DRAWING

[0124] The invention is being illustrated further below in connection with a few examples and embodiment and with reference to the drawings in which:

[0125] FIG. 1 shows a table of pH optimum for a number of enzymes.

[0126] FIG. 2a illustrates a system for determining activity of a target enzyme of a sample.

[0127] FIG. 2b illustrates a system for determining activity of a target enzyme of a grain material.

[0128] FIG. 3a illustrates a standard curve associated to a preselected extraction and digestion procedure.

[0129] FIG. 3b illustrates a further standard curve associated to a preselected extraction and digestion procedure.

[0130] FIG. 4a illustrates data points for five standard curves, each associated to respective preselected extraction times for extracting target enzyme from a grain material.

[0131] FIG. 4b illustrates five standard curves for five different selected extracting times.

[0132] 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.

[0133] FIG. 5b show standard curves of concentration as a function of absorbance for a number of incubation times.

[0134] FIG. 6 shows determined absorbance parameter values as a function of the Ring trial WK values obtained in example 2 an example 3.

[0135] FIG. 7 shows determined absorbance parameter values as a function of the Ring trial WK values obtained in example 4.

[0136] FIG. 8 shows determined absorbance parameter values as a function of the Ring trial WK values obtained in example 5.

[0137] FIG. 9 shows determined absorbance parameter values as a function of the Ring trial WK values obtained in example 6.

[0138] FIG. 10 shows determined absorbance parameter values as a function of the DU values obtained in example 7.

[0139] FIGS. 11a and 11b shows determined absorbance parameter values as a function of the BU3/ml and BU3/g values obtained in example 8.

[0140] FIGS. 12a and 12b shows determined absorbance parameter values as a function of the BU3/ml and BU3/g values obtained in example 9.

[0141] 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.

[0142] 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.

[0143] 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

[0144] 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 substrate 11, a cuvette 13, a pipette 14, a reader 15 and a tablet 16, such as a smartphone.

[0145] 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.

[0146] 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.

[0147] FIG. 2 illustrates a system for determining activity of a target enzyme of a grain material.

[0148] 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.

[0149] 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.

[0150] 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.

[0151] 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.

[0152] It will be observed that the data points for each standard curve are lying on a substantially straight line.

[0153] 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.

[0154] 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.

[0155] 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.

[0156] 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.

[0157] 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.

[0158] 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.

[0159] 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.

[0160] 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.

[0161] 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.

[0162] FIG. 5b show the concentration as a function of absorbance for a number of incubation times.

[0163] In the following examples the terms “Distatic power” and “Ring trial” has the following meaning:

[0164] Diastatic power: The malts ability to break down starches into simpler fermentable sugars during the mashing process. It is measured in Windisch-Kolbach (WK), IoB 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.

[0165] 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

[0166] Constructing a Calibration Curve for Determining Alpha-Amylase Activity in Barley Malt Samples

[0167] 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.

[0168] 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 known alpha-amylase known beta-amylase sample # activity (DU) 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

[0169] 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.

[0170] A fluid extract reference sample is obtained from each barley malt reference sample using a preselected extraction procedure.

[0171] The preselected extraction procedure is as follows: [0172] The samples are milled by use of a lab mill, e.g. Bühler Miag Disc Mill [0173] 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.

[0174] The composition of the liquid extraction buffer was prepared according to the following scheme:

[0175] 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)*2H.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.

[0176] The extraction was performed at room temperature (21° C.).

[0177] 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.

[0178] 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.

[0179] 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.

[0180] 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.

[0181] 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.

[0182] 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.

[0183] Measurement

[0184] 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.

[0185] 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.

[0186] Thereafter the respective absorbance parameter is plotted as a function of alpha amylase activity (DU).

TABLE-US-00002 Barley known alpha- absorbance (# transmitted reference amylase light intensity − blind transmitted sample # activity (DU) 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

[0187] The absorbance is determined according to the formula:

[00001]$A={\log }_{10}\frac{{\Phi }_e^i}{{\Phi }_e^t}=-{\log }_{10}T,$

[0188] where [0189] Φ.sub.e.sup.t is the radiant flux transmitted by that material; [0190] Φ.sub.e.sup.j is the radiant flax received by that material, [0191] T=Φ.sub.e.sup.t/Φ.sub.e.sup.i is the transmittance of that material.

Example 2

[0192] Ring Trial Validation—Extraction Time

[0193] Ring trial validations using five different milled malt samples having different WK values were performed.

[0194] 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

[0195] The samples were tested according to the following protocol [0196] Weigh 200 mg of milled malt. [0197] Extraction with extraction buffer for 10 minutes at 35° C. [0198] Syringe filtration with 0.45 μm filter. Collect the filtrate. [0199] Incubation of 250 μL filtrate with substrate for an incubation time at 35° C. [0200] Stop the enzymatic cleaving by adding a stopping reagent [0201] Read the resulting liquid at 410 nm in a spectrophotometer.

[0202] 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.

[0203] 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.

[0204] The incubation time used was 7 minutes.

[0205] The stopping agent used was an alkaline sodium carbonate solution.

[0206] The spectrophotometer used was the spectrometer sold by Glycospot under the trade name SIRIUS™.

[0207] 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.

[0208] Each test was performed in triplicate and the average absorbance parameter values were determined.

[0209] Thereafter the respective absorbance parameter values were plotted as a function of the Ring trial WK values. The plot is shown in FIG. 6.

[0210] 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

[0211] Reference Curve for Barley Malt—Diastatic Power Range 30-500 WK.

[0212] The test results obtained in example 2 and shown in FIG. 8 were applied as reference curve for barley malt.

[0213] 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

[0214] Ring Trial Validation—Incubation Time

[0215] Ring trial validations using three different milled malt samples having different WK values were performed.

[0216] 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.

[0217] Each sample was tested with three different incubation times, namely 3 minutes, 5 minutes and 7 minutes respectively.

[0218] Each test was performed in triplicate and the average absorbance parameter values were determined.

[0219] Thereafter the respective absorbance parameter values were plotted as a function of the Ring trial WK values. The plot is shown in FIG. 7.

[0220] The results show a very good correlation with the ring trial samples for each of the three incubation times.

[0221] 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

[0222] Preparing Reference Curve for Barley Malt—Diastatic Power Range 500-100 WK.

[0223] 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

[0224] 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. [0225] 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.

[0226] Each test was performed in triplicate and the average absorbance parameter values were determined.

[0227] Thereafter the respective absorbance parameter values were plotted as a function of the Ring trial WK values. The plot is shown in FIG. 8.

[0228] 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

[0229] Preparing Reference Curve for Wheat Malt—Diastatic Power Range 200-500 WK.

[0230] 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

[0231] 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.

[0232] The extraction buffer was as in example 2 but the amount applied was 50 mL together with 70 mg Dithiothreitol/sample.

[0233] Each test was performed in triplicate and the average absorbance parameter values were determined.

[0234] Thereafter the respective absorbance parameter values were plotted as a function of the Ring trial WK values. The plot is shown in FIG. 9.

[0235] 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

[0236] Preparing Reference Curve for Alpha Amylase Activity for Barley Malt (10-100 DU)

[0237] Three different samples of barley malt with known DU values were subjected to an extraction according to the following extraction protocol: [0238] Weigh 200 mg of milled malt. [0239] Extraction with extraction buffer for 10 minutes at 35° C. [0240] Syringe filtration with 0.45 μm filter. Collect the filtrate.

[0241] 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 Dilution ratio (always mL buffer 3 - pipette 250 μL of the Known and Sample dilution step extraction) calculated-DU Sample A 45 181 10.4 3 - 51 9 37 51 DU 4.5 19 99.3 Sample B 36 145 10.2 6 - 40 12 49 30.2 DU 9 37 40.0 Sample C 27 109 22.3 10 - 65.6 9 37 65.6 DU 7 29 83.7

[0242] The samples were tested according to the following protocol: [0243] Incubation of 250 μL filtrate with substrate for an incubation time at 35° C. [0244] Stop the enzymatic cleaving by adding a stopping reagent [0245] Read the resulting liquid at 410 nm in a spectrophotometer.

[0246] The buffer used for the dilution series was the maleic acid based extraction buffer described in example 1.

[0247] 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.

[0248] The incubation time used was 7 minutes.

[0249] The stopping agent used was an alkaline sodium carbonate solution.

[0250] The spectrophotometer used was the spectrometer sold by Glycospot under the trade name SIRIUS™.

[0251] 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.

[0252] Each test was performed in triplicate and the average absorbance parameter values were determined.

[0253] Thereafter the respective absorbance parameter values were plotted as a function of the DU values. The plot is shown in FIG. 10.

[0254] 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

[0255] Preparing Reference Curve for Beta Amylase Activity in Malt in Betamyl-3 Units BU3

[0256] Betamyl-3 unit is the typical unit for beta amylase activity.

[0257] A single sample of barley malt with known BU3 value was subjected to an extraction according to the following extraction protocol: [0258] Weigh 200 mg of milled barley malt. [0259] Extraction with extraction buffer for 10 minutes at 35° C. [0260] Syringe filtration with 0.45 μm filter. Collect the filtrate.

[0261] The sample were subjected to a dilution series to provide in total 6 diluted samples.

[0262] Each of the 6 dilution samples was tested according to the following protocol: [0263] Incubation of 250 μL filtrate with substrate for an incubation time at 35° C. [0264] Stop the enzymatic cleaving by adding a stopping reagent [0265] Read the resulting liquid at 410 nm in a spectrophotometer.

[0266] The buffer used for the dilution series was the maleic acid based extraction buffer described in example 1.

[0267] 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.

[0268] The incubation time used was 7 minutes.

[0269] The stopping agent used was an alkaline sodium carbonate solution.

[0270] The spectrophotometer used was the spectrometer sold by Glycospot under the trade name SIRIUS™.

[0271] 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.

[0272] Each test was performed in triplicate and the average absorbance parameter values were determined.

[0273] 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.

[0274] 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

[0275] Preparing Reference Curve for Beta Amylase Activity in Malt Extract

[0276] 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.

[0277] 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.

[0278] 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.