WO2006128277A2 - Method for detecting flavour compounds in fermented products using enzymes - Google Patents
Method for detecting flavour compounds in fermented products using enzymes Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
Definitions
- the present invention relates to the brewing industry sector, and more particularly to the detection of flavors in the fermentation and/or brewing industry.
- flavour compounds such as ketones, aldehydes and alcohols are produced by yeast
- the purification, identification, expression and characterization of these enzymes allow their use in methods and devices to detect and measure the presence of specific flavour compounds in fermented products.
- yeast consumes nutrients such as amino acids and produces the vicinal diketones diacetyl and 2,3-pentanedione as metabolic by-products.
- the presence of high levels of valine in the wort have been noted to be a factor linked to reduced diacetyl evolution (Petersen et al., Journal of the American Society of Brewing Chemists 62: 131-139, 2004) during fermentation.
- the metabolic processes associated with flavour compound production have not been fully elucidated.
- yeast uses to reduce diacetyl in wort.
- One aim of the present invention is to provide particulars of purification, identification, expression and characterization of enzymes responsible for diacetyl reduction in yeast.
- a method of detecting and measuring a flavour compound in a fermented product comprising contacting said product with a flavour-detecting amount of at least one reductase enzyme.
- the flavour compound may be in one embodiment a ketone, an aldehyde or an alcohol.
- the reductase enzyme can be an oxidoreductase enzyme, such as one selected from the group consisting of an aldehyde reductase enzyme, a keto reductase enzyme, an acetyl reductase enzyme, a primary aminoreductase enzyme, a secondary aminoreductase enzyme, and an NADPH-dependant oxidoreductase enzyme (including any modified derivatives of these enzymes).
- an aldehyde reductase enzyme such as one selected from the group consisting of an aldehyde reductase enzyme, a keto reductase enzyme, an acetyl reductase enzyme, a primary aminoreductase enzyme, a secondary aminoreductase enzyme, and an NADPH-dependant oxidoreductase enzyme (including any modified derivatives of these enzymes).
- the reductase enzyme is preferably Old Yellow Enzyme (OYE), and more preferably OYE1 , OYE2 or OYE 3 isoform.
- OYE Old Yellow Enzyme
- the reductase enzyme can be substantially purified.
- the fermented product is preferably a liquid.
- the reductase enzyme can also be produced by yeast cell, such as ale yeast cell or lager yeast cell and such as one that has been genetically modified for enhancing production of said reductase enzyme.
- a method for detecting reduction of diacetyl compound from a fermentation product comprising the administration of at least one reductase enzyme catalyzing the reduction of diacetyl.
- the fermentation product can be a brewing product.
- the reductase enzyme is preferably Old Yellow Enzyme (OYE), and more preferably OYE1 , OYE2 or OYE3 isoform.
- OYE Old Yellow Enzyme
- Fig. 1 illustrates a silver stained SDS-PAGE gel showing a purified 45kDa diacetyl reducing protein (Old Yellow Enzyme) along with co-purified 3OkDa protein with no suspected diacetyl reductase activity (2) compared to a ladder (1 );
- Fig. 2 illustrates an ethidium bromide stained agarose gel with PCR amplified gene probes for ADH1 (1 ,5), OYE1 (2,6), OYE2 (3,7) and OYE3 (4,8) in ale (5-8) and lager yeast (1-4) species;
- Fig. 3 illustrates reaction velocities for diacetyl assays using yeast D- arabinose dehydrogenase enzyme
- Fig. 4 illustrates examples of diacetyl assays in filtered beer samples using protein cascade mechanism
- Fig. 5 illustrates a process flow chart for determining diacetyl concentrations in beer.
- enzymes that can be used in any method or device that involves a coupled reaction and measurement of the consumption of a co-factor (including, but not limited to, a biosensor, test kit, or plate reader system).
- Diacetyl (a ketone) is one such flavour compound used herein to exemplify the present invention.
- the present disclosure is thus expandable to other flavour compound and is thus not limited only to enzymes for detecting diacetyl.
- ale and lager yeast cultures obtained from Molson Breweries (Montreal, Qc, Canada) were collected at the end of fermentation. All protein purification procedures were performed at 4 0 C. Ale and lager yeast cell pellets (5Og) were washed 3 times, resuspended in cell lysis buffer and ruptured using glass beads. Proteins were differentially precipitated by progressive addition of ammonium sulphate at 20, 40, 60 and 80% of saturation. The protein pellets were resuspended in buffer and assayed for activity as described by Heidlas and Tressl (Heidlas and Tressl, European Journal of Biochemistry 188: 165-174, 1990). Fractions containing the diacetyl reductase activity were further purified using ion exchange and active Red dye chromatographic techniques and assessed for purity by silver stained SDS- PAGE.
- Protein expression was performed in low phosphate induction media (Craig et al., Proceedings of the National Academy of Sciences 88: 2500-2504, 1991 ) and confirmed by Coomassie stained SDS-PAGE. Proteins were purified by affin ⁇ y chromatography, concentrated and the protein concentration determined spectrophotometrically (Gill and von Hippel, Analytical Biochemistry 182: 319, 1989).
- Reaction conditions consisted of an appropriate amount of enzyme and diacetyl (dependant on the K m of the enzyme), 200 ⁇ M NADPH and buffer at pH 7 in a 1 ml cuvette. Enzyme kinetic parameters (K m , k cat ) were determined using Hanes plot analysis and varying quantities of substrates. Purification of diacetyl reductase from brewing yeast
- OYE3 displayed the poorest catalytic turnover rates, with the exception of the unnatural phenylglyoxal substrate. It was not possible to obtain catalytic information for OYE3 with low, physiological diacetyl and pentanedione concentrations. Catalytic information for OYE 1 and OYE3 with physiological pentanedione concentrations was also undeterminable. Phenylglyoxal yielded the lowest K m values for all three OYE isoforms, followed by diacetyl and pentanedione respectively.
- OYE 1 showed the greatest catalytic ability with diacetyl, while OYE2 was able to best utilize 2,3- pentanedione. OYE3 activity was indeterminable with both substrates at very low concentrations.
- OYE3 activity was indeterminable with both substrates at very low concentrations.
- OYE1 isoforms of Old Yellow Enzyme with OYE1 being unique to lager yeast species (S. pastorian ⁇ s I S. carisbergensis etc.) while OYE2 and OYE3 are found in both lager yeast and ale yeast (S. cerevisiae).
- OYE 1 was found to have the best affinity for diacetyl at low concentrations, while OYE2 had the best affinity for pentanedione at low concentrations.
- OYE3 behaved poorly with both substrates and it was not possible to determine rates when low concentrations were used.
- OYE1 has the largest k ⁇ t for all three substrates tested, making it the most efficient enzyme of the three isoforms. It is likely to be the most efficient catalyst of diacetyl reduction at physiological concentrations. While OYE2 is capable of reducing diacetyl at physiological conditions it appears to be more efficient with 2,3-pentanedione. Given that these enzymes have been shown to catalyze reactions at very low substrate concentrations, it is suggested that they are responsible for diacetyl reduction in brewing yeast.
- Beer samples were filtered through a 0.45 ⁇ M filter in series with a solid phase extraction column to reduce background noise due to the high absorbance properties of the beer matrix.
- ⁇ -Acetolactate conversion to diacetyl was catalyzed by the addition of a mixture of FeSO 4 and FeCb and heating of the sample for a predetermined period of time to mediate decarboxylation
- Diacetyl levels in beer samples were determined by adding NADPH to a final concentration of 200 ⁇ M NADPH plus a specific concentration of recombinant diacetyl reductase enzyme; Old yellow enzyme (Table 2), D- Arabinose Dehydrogenase (Table 3). The reduction of diacetyl to acetoin was monitored at 365 nm (Fig. 3, Fig. 4). Data gathered was used to determine the reaction velocity at various diacetyl concentrations and used to build a calibration table. The assay procedure is summarized in Figure 5. Table 2
- Tables 2 and 3 give the kinetic parameters for the OYE enzymes and the D-Arabinose dehydrogenase. These OYE show high specificity i.e low K m for diacetyl which is important for determining diacetyl concentrations in the micromolar or ppb range.
- Figures 3 and 4 demonstrate linearity between the activity and the concentration of the substrate. This is important for generating standards curves that will be used to calculate diacetyl concentration in fermentation products.
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Abstract
The present invention relates to a method of detecting and measuring a flavour compound in a fermented product comprising contacting said product with a flavour-detecting amount of at least one reductase enzyme.
Description
METHOD FOR DETECTING FLAVOUR COMPOUNDS IN FERMENTED PRODUCTS USING ENZYMES
TECHNICAL FIELD
[0001] The present invention relates to the brewing industry sector, and more particularly to the detection of flavors in the fermentation and/or brewing industry.
BACKGROUND OF THE INVENTION
[0002] Reduction of important flavour compounds produced by yeast to below taste threshold levels remains a central concern to many brewing operations.
[0003] Because flavour compounds such as ketones, aldehydes and alcohols are produced by yeast, there exist primary enzymes in yeast that have a dominant reaction with specific flavour compounds. The purification, identification, expression and characterization of these enzymes allow their use in methods and devices to detect and measure the presence of specific flavour compounds in fermented products.
[0004] During wort fermentation, yeast consumes nutrients such as amino acids and produces the vicinal diketones diacetyl and 2,3-pentanedione as metabolic by-products. The presence of high levels of valine in the wort have been noted to be a factor linked to reduced diacetyl evolution (Petersen et al., Journal of the American Society of Brewing Chemists 62: 131-139, 2004) during fermentation. However, the metabolic processes associated with flavour compound production have not been fully elucidated. Although several studies have described a number of enzyme activities in partially purified preparations capable of reducing diacetyl (Heidlas and Tressl, European Journal of Biochemistry 188: 165-174, 1990; Bamforth and Kanauchi, Journal of the Institute of Brewing 110: 83-93, 2004), none of the enzymes associated with the reductase activity have been positively identified.
[0005] In beer fermentation, two scenarios are commonly observed. In the first situation, the yeast produces high levels of diacetyl, which is then gradually reduced to diacetyl specification (commonly 50 parts per billion (ppb)) well after terminal gravity is reached. In the second situation, yeast produces lower levels of diacetyl and diacetyl specification is attained before terminal gravity is reached. In the first situation, terminal gravity is reached a day or two before diacetyl levels are reduced to specification. In certain cases, the time required to deplete diacetyl can account for as much as 20% of the total fermentation time. This lag in the production stream represents a significant financial cost. An understanding of the enzymatic mechanisms involved in diacetyl reduction could lead to new techniques to selectively measure and control diacetyl production and subsequent reduction while avoiding genetic manipulation of yeast strains.
[0006] Currently there is little known about the metabolic mechanisms yeast uses to reduce diacetyl in wort.
[0007] It would be highly desirable to be provided with identified and characterized enzymes exhibiting diacetyl reductase activity, as an initial step towards developing improved fermentation control techniques.
[0008] It would be highly desirable to be provided with characterization of such enzymes to be used in yeast brewing operations to detect flavour compounds in fermented products
SUMMARY OF THE INVENTION
[0009] One aim of the present invention is to provide particulars of purification, identification, expression and characterization of enzymes responsible for diacetyl reduction in yeast.
[0010] In accordance with the present invention there is provided a method of detecting and measuring a flavour compound in a fermented product comprising contacting said product with a flavour-detecting amount of at least one reductase enzyme.
[0011] The flavour compound may be in one embodiment a ketone, an aldehyde or an alcohol.
[0012] The reductase enzyme can be an oxidoreductase enzyme, such as one selected from the group consisting of an aldehyde reductase enzyme, a keto reductase enzyme, an acetyl reductase enzyme, a primary aminoreductase enzyme, a secondary aminoreductase enzyme, and an NADPH-dependant oxidoreductase enzyme (including any modified derivatives of these enzymes).
[0013] In one embodiment, the reductase enzyme is preferably Old Yellow Enzyme (OYE), and more preferably OYE1 , OYE2 or OYE 3 isoform.
[0014] The reductase enzyme can be substantially purified.
[0015] The fermented product is preferably a liquid.
[0016] The reductase enzyme can also be produced by yeast cell, such as ale yeast cell or lager yeast cell and such as one that has been genetically modified for enhancing production of said reductase enzyme.
[0017] In another embodiment, there is provided a method for detecting reduction of diacetyl compound from a fermentation product comprising the administration of at least one reductase enzyme catalyzing the reduction of diacetyl.
[0018] The fermentation product can be a brewing product.
[0019] Further, the reductase enzyme is preferably Old Yellow Enzyme (OYE), and more preferably OYE1 , OYE2 or OYE3 isoform.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] Fig. 1 illustrates a silver stained SDS-PAGE gel showing a purified 45kDa diacetyl reducing protein (Old Yellow Enzyme) along with co-purified 3OkDa protein with no suspected diacetyl reductase activity (2) compared to a ladder (1 );
[0021] Fig. 2 illustrates an ethidium bromide stained agarose gel with PCR amplified gene probes for ADH1 (1 ,5), OYE1 (2,6), OYE2 (3,7) and OYE3 (4,8) in ale (5-8) and lager yeast (1-4) species;
[0022] Fig. 3 illustrates reaction velocities for diacetyl assays using yeast D- arabinose dehydrogenase enzyme;
[0023] Fig. 4 illustrates examples of diacetyl assays in filtered beer samples using protein cascade mechanism; and
[0024] Fig. 5 illustrates a process flow chart for determining diacetyl concentrations in beer.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
[0025] In accordance with the present invention, there is thus provided enzymes that can be used in any method or device that involves a coupled reaction and measurement of the consumption of a co-factor (including, but not limited to, a biosensor, test kit, or plate reader system).
[0026] Diacetyl (a ketone) is one such flavour compound used herein to exemplify the present invention. The present disclosure is thus expandable to other flavour compound and is thus not limited only to enzymes for detecting diacetyl.
Purification of diacetyl reductases
[0027] In accordance with the present invention, ale and lager yeast cultures obtained from Molson Breweries (Montreal, Qc, Canada) were collected at the end of fermentation. All protein purification procedures were performed at 40C. Ale and lager yeast cell pellets (5Og) were washed 3 times, resuspended in cell lysis buffer and ruptured using glass beads. Proteins were differentially precipitated by progressive addition of ammonium sulphate at 20, 40, 60 and 80% of saturation. The protein pellets were resuspended in buffer and assayed for activity as described by Heidlas and Tressl (Heidlas and Tressl, European Journal of Biochemistry 188: 165-174, 1990). Fractions containing the diacetyl reductase activity were further purified using ion exchange and active Red dye
chromatographic techniques and assessed for purity by silver stained SDS- PAGE.
Protein identification
[0028] Purified proteins were treated with trypsin and the digested mixture was then analyzed using Matrix Assisted Laser Desorption/lonisation Time-of- flight (MALDI-TOF) mass spectrometry. The mass fingerprint was used to search the S. cerevisiae database using the Mascot™ search engine. Protein identifications were further confirmed by fragmentation sequence analysis using electrospray (ES) tandem mass spectrometry.
Protein expression and purification
[0029] The open reading frames for three proteins were amplified by polymerase chain reaction (PCR) using Platinum® Pfx polymerase (Invitrogen, ON, Canada) using sequence specific oligonucleotide primers (Table 1 ) (Qiagen, ON, Canada) and the sequence integrity confirmed by automated DNA sequence analysis. The PCR products were cloned into pBAce expression plasmid (Craig et al., Proceedings of the National Academy of Sciences 88: 2500-2504, 1991 ) and used to transform E. coli ER2566 cells (New England Biolabs, MA, USA). Protein expression was performed in low phosphate induction media (Craig et al., Proceedings of the National Academy of Sciences 88: 2500-2504, 1991 ) and confirmed by Coomassie stained SDS-PAGE. Proteins were purified by affinϋy chromatography, concentrated and the protein concentration determined spectrophotometrically (Gill and von Hippel, Analytical Biochemistry 182: 319, 1989).
Kinetic analysis
[0030] Reaction conditions consisted of an appropriate amount of enzyme and diacetyl (dependant on the Km of the enzyme), 200 μM NADPH and buffer at pH 7 in a 1 ml cuvette. Enzyme kinetic parameters (Km, kcat) were determined using Hanes plot analysis and varying quantities of substrates.
Purification of diacetyl reductase from brewing yeast
[0031] Using the method described above, proteins showing diacetyl reductase activity were successfully purified from ale and lager yeasts using conventional chromatographic techniques that included ion exchange and dye affinity chromatography. A preparation containing diacetyl reductase activity was obtained. Analysis of this purified protein preparation on silver-stained SDS PAGE revealed two bands with molecular weights of 45kDa and 3OkDa (Fig. 1 ). Mass fingerprint analysis of this fraction using MALDI-TOF identified Old Yellow Enzyme (OYE, EC 1.6.99.1 ), an NADPH requiring enzyme with unknown function, with a score of 61. The identity of this protein was further confirmed by de novo sequence analysis using tandem mass spectrometry. The smaller 3OkDa band appeared to be a non-catalytic protein with similar physical properties, leading to co-elution with OYE proteins. Searches of the Saccharomyces Genome Database using the OYE2 protein sequence identified three isoforms of this enzyme. PCR analysis of genomic DNA isolated from ale or lager yeast using primer-pairs specific for OYE1 , OYE2 and OYE3 revealed a differential distribution of these genes in the two yeast strains (Table 1 ). OYE1 was found exclusively in lager yeast strains, while OYE2 and OYE3 isoforms were found in ale yeast and in lager yeast (Fig. 2). As a control for template integrity, the alcohol dehydrogenase (ADH) gene was used. A strong band for ADH was obtained for both ale and lager yeast.
Table 1 Primer sequences used for gene amplification of Old Yellow Enzyme isoforms.
Recombinant diacetyl reductase and kinetic analysis
[0033] To validate the conjecture that OYE enzymes could reduce diacetyl, all three isoforms were expressed in an E. coli expression system and the purified recombinant proteins were assessed for enzymatic activity. All three isoforms of OYE were expressed in high levels as soluble proteins and kinetic analysis revealed that the OYE enzymes were capable of utilizing diacetyl, pentanedione and phenylglyoxal. Of note, however, was the apparent biphasic kinetic behaviour exhibited by all three OYE isoforms.
Under steady-state conditions, OYE1 exhibited the greatest turnover rates (kcat) with all three substrates followed by OYE2 and OYE3. The biphasic characteristic of these enzymes resulted in two Michaelis-Menton constants (Km), one at the low micromolar (μM), and the other at the millimolar (mM) concentration level. High-end Km values ranged from 0.5 - 14 mM for all three substrates, whereas the low-end (physiological concentration) Km values ranged from 3 - 180 μM (258 ppb and up for diacetyl).
[0034] OYE3 displayed the poorest catalytic turnover rates, with the exception of the unnatural phenylglyoxal substrate. It was not possible to obtain catalytic information for OYE3 with low, physiological diacetyl and pentanedione concentrations. Catalytic information for OYE 1 and OYE3 with physiological pentanedione concentrations was also undeterminable. Phenylglyoxal yielded the lowest Km values for all three OYE isoforms, followed by diacetyl and pentanedione respectively.
[0035] At lower, physiological concentrations, OYE 1 showed the greatest catalytic ability with diacetyl, while OYE2 was able to best utilize 2,3- pentanedione. OYE3 activity was indeterminable with both substrates at very low concentrations.
[0036] To elucidate the enzymatic metabolic systems used in the reduction of diacetyl, conventional chromatography techniques were used to identify enzymes that are capable of catalyzing diacetyl and pentanedione reduction. Genetic queries yielded three isoforms of Old Yellow Enzyme with OYE1 being unique to lager yeast species (S. pastorianυs I S. carisbergensis etc.) while OYE2 and OYE3 are found in both lager yeast and ale yeast (S. cerevisiae).
[0037] All three isoforms were successfully expressed in their active forms using recombinant methods. OYE 1 was found to have the best affinity for diacetyl at low concentrations, while OYE2 had the best affinity for pentanedione at low concentrations. OYE3 behaved poorly with both substrates and it was not possible to determine rates when low concentrations were used.
[0038] Higher, non-physiological, Km values were obtained for all three enzymes with all three substrates. OYE 1 had the lowest Km for diacetyl as well as the highest
OYE 1 was the most efficient enzyme catalyst of the three isoforms with higher, mM (100ppm+) concentrations of diacetyl and 2,3- pentanedione.
[0039] Although it would appear that the utility of OYE3 is questionable, the kcat is only slightly smaller than that of OYE2 for pentanedione, suggesting that OYE2 and OYE3 catalyze higher concentrations of 2,3-pentanedione with almost the same efficiency. Results with physiological concentrations suggest that OYE2 is responsible for 2,3-pentanedione reduction in actually brewing conditions.
[0040] All three isoforms yielded low Km values with phenylglyoxal, which were not unexpected. Previous research (Schopfer and Massey, Old Yellow Enzyme, in Mechanism of Enzyme Action (Kuby, S. A., Ed.) pp 247-269, CRC Press, Boca Raton, Florida, 1991 ; Vaz et al., Biochemistry 34:424 6-4256, 1995) has shown tight binding of aromatic ring compounds. Although phenylglyoxal is not a naturally occurring substrate, it possesses both the aromatic ring structure as well as a diketone side chain. The results disclosed in the present invention indicate that OYE enzymes catalyze the reduction of this
compound quite efficiently although not as efficiently as some other aromatic compounds.
[0041] Overall, OYE1 has the largest k∞t for all three substrates tested, making it the most efficient enzyme of the three isoforms. It is likely to be the most efficient catalyst of diacetyl reduction at physiological concentrations. While OYE2 is capable of reducing diacetyl at physiological conditions it appears to be more efficient with 2,3-pentanedione. Given that these enzymes have been shown to catalyze reactions at very low substrate concentrations, it is suggested that they are responsible for diacetyl reduction in brewing yeast.
[0042] The present invention would be readily understood by referring to the following examples which are given to illustrate the invention rather than to limits its scope .
Example 1
Sample preparation and enzymatic assay for diacetyl detection in fermentation streams.
[0043] Beer samples were filtered through a 0.45 μM filter in series with a solid phase extraction column to reduce background noise due to the high absorbance properties of the beer matrix. σ-Acetolactate conversion to diacetyl was catalyzed by the addition of a mixture of FeSO4 and FeCb and heating of the sample for a predetermined period of time to mediate decarboxylation
[0044] Diacetyl levels in beer samples were determined by adding NADPH to a final concentration of 200 μM NADPH plus a specific concentration of recombinant diacetyl reductase enzyme; Old yellow enzyme (Table 2), D- Arabinose Dehydrogenase (Table 3). The reduction of diacetyl to acetoin was monitored at 365 nm (Fig. 3, Fig. 4). Data gathered was used to determine the reaction velocity at various diacetyl concentrations and used to build a calibration table. The assay procedure is summarized in Figure 5.
Table 2
Kinetic properties of Old Yellow Enzyme lsoforms with dicarbonyl substrates diacetyl and pentanedione.
Diacetyl Pentanedione
Km1 kcati Km2 kcat2 Km1 kcati Km2 kcat2
Enzyme
(μM) (min'1) (mM) (min"1) (μM) (min-1) (mM) (min-1)
OYE1 2.40 0.88 3.20 27.5 n/a n/a 12.4 81.2
OYE2 2.00 0.42 5.40 7.10 224 2.20 8.40 33.7
OYE3 n/a n/a 10.8 2.1 n/a n/a 14.3 21.5
Table 3
Kinetic properties of D-Arabinose dehydrogenase with dicarbonyl substrates diacetyl, pentanedione and methylglyoxal.
Km (mM) kcat (s-1) kcat/Km
Methylglyoxal 14.3 4.4 0.30
Diacetyl 7.7 6.9 0.9
Pentanedione 4.2 5.9 1.41
NADPH 0.014 3.7 257
[0045] These results demonstrate the level of sensitivity and efficacy of these enzymes activity on these substrates (diacetyl, pentanedione and methylglyoxal.
[0046] Tables 2 and 3 give the kinetic parameters for the OYE enzymes and the D-Arabinose dehydrogenase. These OYE show high specificity i.e low Km for diacetyl which is important for determining diacetyl concentrations in the micromolar or ppb range.
[0047] Figures 3 and 4 demonstrate linearity between the activity and the concentration of the substrate. This is important for generating standards curves that will be used to calculate diacetyl concentration in fermentation products.
[0048] While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or
adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth, and as follows in the scope of the appended claims.
Claims
1. A method of detecting and measuring a flavour compound in a fermented product comprising contacting said product with a flavour- detecting amount of at least one reductase enzyme.
2. The method of claim 1 , wherein said flavour compound is a ketone, an aldehyde or an alcohol.
3. The method of claim 1 or 2, wherein said reductase enzyme is an oxidoreductase enzyme.
4. The method of any one of claims 1-3, wherein said oxidoreductase enzyme is selected from the group consisting of an aldehyde reductase enzyme, a keto reductase enzyme, an acetyl reductase enzyme, a primary aminoreductase enzyme, a secondary aminoreductase enzyme, and an NADPH-dependant oxidoreductase enzyme.
5. The method of claim 4, wherein said reductase enzyme is Old Yellow Enzyme (OYE).
6. The method of claim 5, wherein said OYE is OYE 1 isoform.
7. The method of claim 5, wherein said OYE is OYE2 isoform.
8. The method of claim 5, wherein said OYE is 0YE3 isoform.
9. The method of claim 1 , wherein said reductase enzyme is substantially purified.
10. The method of claim 1 , wherein said fermented product is a liquid.
11. The method of claim 1 , wherein said reductase enzyme is produced by a yeast cell.
12. The method of claim 11 , wherein said yeast cell is a lager yeast cell.
13. The method of claim 11 , wherein said yeast cell is an ale yeast cell.
14. The method of claim 1 , wherein said yeast cell has been genetically modified for enhancing production of said reductase enzyme.
15. A method for detecting reduction of diacetyl compound from a fermentation product comprising the administration of at least one reductase enzyme catalyzing the reduction of diacetyl.
16. The method of claim 15, wherein the fermentation product is a brewing product.
17. The method of any of claim 15 or 16, wherein said reductase enzyme is Old Yellow Enzyme (OYE).
18. The method of claim 17, wherein said OYE is OYE1 isoform.
19. The method of claim 17, wherein said OYE is OYE2 isoform.
20. The method of claim 17, wherein said OYE is OYE3 isoform.
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Cited By (4)
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| EP2123767A1 (en) * | 2008-05-20 | 2009-11-25 | DSM IP Assets B.V. | Preparation of epsilon-caprolactam via lysine cyclisation |
| WO2009142490A1 (en) * | 2008-05-20 | 2009-11-26 | Dsm Ip Assets B.V. | Preparation of epsilon-caprolactam from (z)-6,7-dihydro-1h-azepin-2(5h)-one |
| EP2382308A2 (en) * | 2008-12-25 | 2011-11-02 | Codexis, Inc. | Enone reductases |
| WO2020095008A1 (en) * | 2018-11-08 | 2020-05-14 | Institut Des Sciences Et Industries Du Vivant Et De L'environnement - Agroparistech | BIOCATALYTIC METHOD FOR PRODUCING 2H-HBO AND β-SUBSTITUTED ANALOGUES FROM LGO USING A CYCLOHEXANONE MONOOXYGENASE |
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