Co-culture for efficient production of fermented beverages

Abstract

The present invention relates to a method for producing a fermented beverage using at least two fermenting yeast strains, to a fermented beverage which may be produced by the method of the invention, and to the use of at least two fermenting yeast strains for the production of a fermented beverage.

Description

[Technical field]

[0001] The present invention is in the field of microbiology. In particular, the present invention relates to the use of yeast strains with different substrate preferences and / or consumption capacities for sugar to produce a fermented product, preferably beer. [Background technology]

[0002] The first step in the fermentation of brewery wort sugars is the transport of the sugars across the cell membrane. The efficiency and completeness of wort sugar assimilation depends on the abundance and specificity of transport proteins for the fermentable sugars glucose, fructose, maltose and maltotriose, but also on the metabolism of the sugars after they enter the cell. The conversion of sugar mixtures by microorganisms is usually regulated so that the cell allocates its intracellular resources for the conversion of different substrates in a sequential manner, starting with the most easily converted sugars. During the fermentation of brewery wort, especially at high gravity, this reduces the degree of fermentation and causes the presence of significant concentrations of residual sugars. This is at least partly due to the fact that the alcohol produced prevents the fermentation of the remaining sugars, which are generally more difficult to convert.

[0003] This problem has been recognized in the process of producing alcohol. For example, published WO 2013 / 181496 proposes using a combination of different yeast strains. Since the goal of WO 2013 / 181496 is the production of alcohol, such a combination includes one or more high alcohol tolerance yeasts and one or more maltotriose positive yeasts. Furthermore, preferred substrates for fermentation include starches such as potato or wheat to which no hops are added. The preferred fermentation temperature is between 30°C and 40°C. These features cannot be used for the production of fermented beverages such as beer, especially lager beer.

[0004] There is therefore a need to develop strategies to at least partially overcome the residual sugar concentrations in processes for producing fermented beverages such as beer, in particular lager beer, which preferably allow high gravity wort fermentation. Summary of the Invention

[0005] In nature in general, but in microbiology in particular, trade-offs between nutrient preferences have resulted in organisms specialized for wort sugar assimilation. Here, a method is provided that exploits the diversity in specialization. Conversion of a substrate mixture by a consortium of specialist strains with different substrate specificities, for example preferring maltose or maltotriose, will improve the mixed sugar fermentation kinetics compared to the application of a single strain, such as a generalist strain. The consortium of specialist strains at least partially improves the fermentation degree, resulting in an increase in ethanol yield, especially when fermenting high gravity wort. In contrast to the disclosure of WO 2013 / 181496, such combinations, surprisingly, do not necessarily include one or more high alcohol tolerance yeasts.

[0006] The present invention therefore provides a method for producing a fermented beverage comprising the steps of providing a wort, adding hops and at least two fermenting yeast strains to the wort, such that said at least two fermenting yeast strains differ in substrate specificity for sugars in the wort, in particular for glucose, maltose, fructose and / or maltotriose, and incubating the wort with the at least two fermenting yeast strains for a period of time, thereby producing a fermented beverage. The at least two fermenting yeast strains may optionally be removed from the fermented wort at the end of the incubation period.

[0007] The method of the present invention allows for surprisingly improved sugar fermentation kinetics, such that the desired final gravity is reached at an earlier time point, compared to conventional fermentation using a single non-specialist yeast strain. Those skilled in the art will appreciate that this reduction in time is achieved when a particular wort is fermented with at least two fermenting yeast strains, compared to a single yeast strain, such as a non-specialist yeast strain, even if all other circumstances, such as timing, inoculation amount, fermentation temperature, etc., are substantially identical.

[0008] Additionally, the methods of the present invention allow for the fermentation of high gravity wort or extra high gravity wort having a gravity greater than Plateau 16 degrees, such as Plateau 17 degrees, Plateau 18 degrees, Plateau 19 degrees, Plateau 20 degrees, Plateau 21 degrees, Plateau 22 degrees, or Plateau 23 degrees. This may result in the production of a fermented beverage with a high alcohol content, such as greater than 5% alcohol by volume (ABV), greater than 6% ABV, or greater than 7% ABV.

[0009] Such at least two fermenting yeast strains may be added to the wort simultaneously or sequentially.

[0010] In a preferred method of the present invention, fermentation of high gravity wort, such as wort having a plateau greater than 16 degrees, including plateau 17 degrees, plateau 18 degrees, plateau 19 degrees, or plateau 20 degrees, results in a fermented beverage with a final gravity of less than 2.5 degrees plateau within a short period of time, such as within 15 days of fermenting a plateau 16-18 degree wort.

[0011] In a preferred method of the invention, said at least two fermenting yeast strains are Saccharomyces cerevisiae, S. pastorianus (S. carlsbergensis), S. eubayanus yeast strains, and / or mixtures or hybrids thereof. Such at least two fermenting yeast strains may be strains from the same yeast species, such as a single hybrid species, or may be from two or more yeast species, provided that these strains have different substrate specificities for sugars, in particular for glucose, fructose, maltose, and / or maltotriose.

[0012] In a preferred method of the invention, at least two fermenting yeast strains do not produce 4-vinylguaiacol, for example by inactivation of the putative flavin prenyltransferase (PAD1) gene and / or the ferulic acid decarboxylase (FDC1) gene.

[0013] In a preferred method of the present invention, the fermentation is carried out at a temperature of 6 to 25°C, preferably 8 to 15°C.

[0014] In a preferred method of the invention, the fermented beverage is beer, preferably lager beer.

[0015] In a preferred method of the invention, the fermenting yeast strain with a preference for a maltose substrate is Saccharomyces pastorianus strain CBS1483.

[0016] In a preferred method of the invention, the fermenting yeast strain with a preference for a maltotriose substrate is S. pastorianus strain CBS1513.

[0017] The present invention further provides a fermented beverage, preferably beer, more preferably lager beer, having a high alcohol content, such as greater than 5% alcohol by volume (ABV), greater than 6% ABV, or greater than 7% ABV, such fermented beverage, preferably beer, more preferably lager beer, preferably produced by the method of the present invention.

[0018] The present invention further provides the use of a combination of at least two fermenting yeast strains differing in their substrate specificity towards sugars in the wort, in particular towards glucose, fructose, maltose and / or maltotriose, for the production of a fermented beverage, preferably beer, most preferably lager beer.

[0019] The at least two fermenting yeast strains may be added to the wort simultaneously or sequentially.

[0020] Such at least two fermenting yeast strains preferably comprise a fermenting yeast strain having a maltose substrate preference, such as Saccharomyces pastorianus strain CBS1483, a fermenting yeast strain having a maltotriose substrate preference, such as S. pastorianus strain CBS1513, or a combination thereof. [Brief description of the drawings]

[0021] [Figure 1] Representative sugar consumption profiles of maltose (circles) and maltotriose (squares) for strain WS34 / 70. Average values ​​from biological triplicates are shown. The points used to calculate the respective consumption rates are indicated by black circles for maltose and black squares for maltotriose. [Diagram 2] Distribution of M2 / M3 rate ratios across all strains tested. Rate ratios for CBS1483, CBS1513, and WS34 / 70 are shown as open circles, open squares, and open triangles, respectively. [Diagram 3] Fermentation of 20° P wort. Gravity (degree plateau) traces (A), maltotriose content (B), maltose content (C), and ethanol production levels during (D) and final (E) fermentation of 20° P wort with WS34 / 70, CBS1513, CBS1483, and a combination of CBS1513 and CBS1483 are shown. [Figure 4]Fermentation of wort at 23° P. Gravity (degree plateau) traces (A), maltotriose content (B) and maltose content (C) during fermentation of wort at 23° P with CBS1513, CBS1483 and a combination of CBS1513 and CBS1483 are shown. [Diagram 5] Comparison of fermentation of wort at 20 degrees P with fermentation of wort at 23 degrees P. Gravity (degree plateau) trajectories (A) and final ethanol production levels (B) are shown for the combinations CBS1513 and CBS1483 shown. [Figure 6] Figure 1. Wort fermentation in co-culture with Saccharomyces strains. A) Plateau profile, B) Maltose profile, C) Maltotriose profile, and D) Final ethanol titer (□) of 23°P wort fermentation of a Saccharomyces cerevisiae strain mixture containing Superstart and Thermosacc strains (■) and a Saccharomyces pastorianus strain mixture containing CBS1483 and CBS1513 strains. Values ​​represent the mean ± mean deviation of data from independent quadruplicate cultures. [Figure 7] CO profiles of wort fermentations with a Saccharomyces cerevisiae strain mixture (■) containing strains Superstart and Thermosacc (Lallemand), and a Saccharomyces pastorianus strain mixture (□) containing strains CBS1483 and CBS1513. Profiles shown are from a single representative experiment from a set of quadruplicates. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] 4.1 Definitions. As used herein, the term "fermented beverage" refers to a beer product produced by fermentation of crops and their products, such as, for example, cereals, rice, grapes and other fruits, nuts and / or exudates from, for example, agave, yucca and cacti. A preferred fermented beverage is beer. A more preferred fermented beverage is lager beer.

[0023] As used herein, the term "gene" refers to any and all cis-acting genomic sequences that ensure that a product encoded by the gene is expressed. Such cis-acting genomic sequences include enhancer and promoter sequences, exon and intron sequences, terminator sequences, etc. Such products may be RNA molecules, such as mRNA molecules or siRNA molecules, and / or proteins.

[0024] As used herein, the term "inactivated gene" refers to a gene that is unable to perform its normal function. For example, for a protein-coding gene, "inactivated" means that the gene expression of the protein is reduced and / or that the gene encodes an inactive protein or encodes a protein with reduced activity. Such inactivation can be due to, for example, a change in the promoter sequence such that the promoter cannot initiate transcription of the gene, a change in the splicing site of an intron that prevents correct splicing of the transcribed pre-mRNA, or a change in the coding region of the gene, thereby making the encoded protein more inactive or inactive. Such inactivation is preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, more preferably at least 90%, more preferably at least 99%, compared to a non-inactivated gene, which means that the protein product of the gene has up to 50%, more preferably up to 40%, more preferably up to 30%, more preferably up to 20%, more preferably up to 10%, more preferably up to 1% of the maximum activity of the wild-type protein. This decreased activity may be due to reduced expression and / or decreased activity of the protein encoded by the inactivated gene compared to the protein encoded by the wild-type, i.e., non-inactivated, gene.

[0025] As used herein, the term "hybrid" or "hybrid yeast" refers to a yeast that is the result of combining the genomes of two yeasts of different varieties or species. A hybrid is preferably the result of sexual mating, i.e., a hybrid yeast is the result of mating two cells of different sexes, e.g., two cells of different mating types, preferably two gametes, also called fusion.

[0026] As used herein, the term "interspecies hybrid" refers to a yeast that is the result of combining the genomes of two organisms of different species or even different genera.

[0027] As used herein, the term "yeast" refers to a eukaryotic, unicellular microorganism classified as a member of the fungal kingdom. Preferred yeasts are those of the Saccharomyces sensu stricto complex, including any hybrids thereof. The Saccharomyces sensu stricto complex currently includes eight different species: Saccharomyces cerevisiae, S. paradoxus, S. uvarum, S. mikatae, S. kudriavzevii, S. arboricola, S. eubayanus, and the recently discovered S. jurei (see Hittinger, 2013. Trends Genet 29: 309-317; Naseeb et al., 2017. Int J Syst Evol Microbiol 67: 2046-2052).

[0028] As used herein, the term "fermenting yeast" refers to yeasts of the Saccharomyces sensu stricto complex, preferably S. cerevisiae or S. eubayanus yeasts, and / or hybrids thereof, such as S. pastorianus, also known as S. carlsbergensis.

[0029] As used herein, the term "sugars" refers to sugars present in wort. These sugars include maltose, maltotriose, glucose, maltotetraose, sucrose, dextrin, and fructose. In a typical wort, maltose accounts for about 40-50% of the sugars, maltotriose and glucose each account for about 5-15%, maltotetraose and sucrose each account for about 4-5%, dextrin accounts for about 20-25%, and fructose accounts for about 2.5%. Of these sugars, maltotetraose and dextrin are considered non-fermentable.

[0030] As used herein, the term "wort" refers to the aqueous liquid extracted from milled grains, including or consisting of barley. Starting wort without added hops may be called "sweetwort" and wort with added hops may be called "bitterwort".

[0031] As used herein, the term "maltose" refers to a disaccharide consisting of two glucose molecules linked via an α-1,4 glycosidic bond.

[0032] As used herein, the term "maltotriose" refers to a trisaccharide consisting of three glucose molecules linked via α-1,4 glycosidic bonds.

[0033] As used herein, the term "maltotetraose" refers to the tetrasaccharide ((2R,3R,4S,5S,6R)-2-[(2R,3S,4R,5R,6R)-6-[(2R,3S,4R,5R,6R)-4,5-dihydroxy-2-(hydroxymethyl)-6-[(2R,3S,4R,5R,6S)-4,5,6-trihydroxy-2-(hydroxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-4,5-dihydroxy-2-(hydroxymethyl)oxan-3-yl]oxy-6-(hydroxymethyl)oxane-3,4,5-triol), which is considered to be non-fermentable.

[0034] As used herein, the term "dextrin" refers to a mixture of polymers of D-glucose units linked by α-(1→4) or α-(1→6) glycosidic bonds. Dextrins are formed during the degradation of starch by a series of α- and β-amylases in the mashing process.

[0035] As used herein, the term "substrate preference" refers to the ability of a yeast strain to transport and metabolize a substrate. In the context of the present application, such substrates are preferably sugars selected from maltose, maltotriose, fructose, and glucose. Yeast can be selected for increased metabolism of a particular substrate, for example maltose, maltotriose, fructose, or glucose, meaning that such a particular substrate is preferentially transported and metabolized, preferably even in the presence of other sugars, when compared to a reference yeast, such as a parent yeast before selection. For example, a yeast strain with a substrate preference for maltotriose transports and metabolizes maltotriose in preference to maltotriose. It has been found that a combination of at least two yeast strains that differ in their substrate preferences is optimal, especially for fermenting high gravity wort. Moreover, such a combination of at least two yeast strains that differ in their substrate preferences enhances the fermentability of the resulting fermented beverage. In other words, such a combination of at least two yeast strains with different substrate preferences resulted in an apparent fermentation degree of at least 0.8 in a fermented beverage in a shorter period of time compared to a single yeast strain, such as a single generalist or standard strain.

[0036] As used herein, the term "maltose preference" refers to a yeast strain that is able to transport and metabolize maltose with a higher preference compared to the parent strain. Such term preferably refers to yeast with a maltose / maltotriose rate ratio of 3 or more, preferably 4-10 or more.

[0037] As used herein, the term "maltotriose preference" refers to a yeast strain that can transport and metabolize maltotriose with a higher preference compared to the parent strain. Such term preferably refers to a yeast having a maltose / maltotriose rate ratio of less than 2, preferably less than 1, for example between 0.01 and 1.

[0038] As used herein, the term "generalist" refers to a yeast strain that has no preference for the conversion of maltose or maltotriose. Such term preferably refers to a yeast having a maltose / maltotriose rate ratio of 1 to 4, such as about 2, preferably 1.6 to 2.2.

[0039] As used herein, the term "maltose rate" refers to the rate of maltose fermentation by a yeast strain. Such rate is preferably determined in the exponential growth phase of the maltose consumption curve. Such rate is preferably determined according to the formula Y t =Y0*e (k1*t ) where k1(gL -1 h -1 ) is the rate constant, and Y t (gL -1 ) is the maltose concentration at time t, and Y0(gL -1 ) is the starting maltose concentration at t=0, and t (hours) is the time, the point in the exponential growth phase of the maltose consumption curve. The constant k1 is a calculated value for a particular strain or combination of strains.

[0040] As used herein, the term "maltotriose rate" refers to the rate of maltotriose fermentation by a yeast strain. Such rate is preferably determined in the exponential growth phase of the maltotriose consumption curve. Such rate is preferably determined according to the formula Y t =Y0*e (k2*t ) where k2(gL -1 h -1 ) is the rate constant, and Y t (gL -1) is the maltotriose concentration at time t, and Y0(gL -1 ) is the starting maltose concentration at t=0, t(hr) is the time, the point in the exponential growth phase of the maltotriose consumption curve. The constant k2 is a calculated value for a particular strain or combination of strains.

[0041] The term "maltose / maltotriose rate ratio" refers to the determined maltose rate divided by the determined maltotriose rate (k1 / k2), resulting in a dimensionless value of the maltose / maltotriose (M2 / M3) rate ratio. The ratio can be maximized to a value of 10. Again, the constant k is a calculated value for a particular strain or combination of strains.

[0042] As used herein in the context of fermentation of alcoholic beverages, the term "gravity" refers to the relative density of a fluid compared to water, which is highly dependent on the sugar content of the fluid. Gravity may be determined by a hydrometer, a refractometer, a pycnometer, or a vibrating U-tube electronic meter, as known to those skilled in the art.

[0043] As used herein, the term "specific gravity" refers to the relative density of a fluid at a reference temperature to the density of water at the reference temperature. This can be measured using either a hydrometer or a refractometer on an appropriate scale.

[0044] As used herein, the term "original extract" refers to the grams of sugar in 100 grams of wort before fermentation, as measured, for example, on a degree plateau, Brix, or dissolved solids scale. It can be measured directly with certain refractometers and hydrometers, or it can be converted from the original gravity.

[0045] As used herein, the term "original gravity" refers to a measure of specific gravity at a given reference temperature before fermentation. It can be measured directly with a specific refractometer and hydrometer, or it can be converted from the original extract.

[0046] As used herein, the term "extract" is a measure of the total fermentable sugars and non-fermentable soluble carbohydrates in the wort, whereby a solution containing a plateau of X degrees of extract has the same density as an aqueous solution containing X grams of sucrose in 100 g of solution.

[0047] As used herein, the term "apparent extract" refers to the portion of the original extract that is present as residual sugars that were not converted to yeast biomass, ethanol, or CO2 during fermentation. It can be expressed interchangeably as degree plateau, Brix, or dissolved solids, and is usually determined by calculating based on the original extract without correcting for the effect of ethanol on density. Apparent extract can be calculated from density, for example, by Analytica-EBC method 9.4 (EBC-European Brewery Convention. 2004. Analytica-EBC. Verlag Hans Carl Getranke-Fachverlag, Nuremberg, Germany).

[0048] As used herein, the term "degree of fermentation" refers to the amount of sugars present in the wort that are converted to CO2 and some other compounds such as alcohols and esters through fermentation. The degree of fermentation refers to the percentage of sugars that are fermented compared to the amount of sugars present at the start, and is preferably expressed in degrees plateau (°P).

[0049] As used herein, the term "apparent fermentation" refers to the relative conversion of sugars present in the starting wort without correcting for the effect of ethanol on density. Apparent fermentation can be calculated by the following formula: ([original extract-apparent extract] / original extract). The term "actual fermentation" refers to the relative conversion of sugars present in the starting wort while correcting for the effect of ethanol on density.

[0050] As used herein, the term "final gravity" refers to a measure of specific gravity at a given reference temperature at the end of fermentation and is directly related to the apparent extract. The gravity measurement can signal the end of fermentation when motion stops. The final gravity can be measured directly with a hydrometer or calculated based on the original extract and the apparent extract.

[0051] As used herein, the term "high gravity wort" refers to wort with a plateau of greater than 14 degrees, preferably up to a plateau of 23 degrees. High gravity wort may be fermented to produce a beer with an alcohol by volume (ABV) of at least 6%.

[0052] 4.2 Yeast strains with different hierarchical substrate preferences. As known to those skilled in the art, yeast strains with a preference for sugars selected from maltose, maltotriose, fructose and glucose can be generated in several ways. For example, such specialists may exist in nature and the isolation of such specialists involves selection on a synthetic growth medium that contains, for example, one of maltose, maltotriose, fructose and glucose as the only carbon source. For example, successive selections on such selective growth media can be performed to select strains that prefer one of maltose, maltotriose, fructose and glucose by lowering the concentration of the preferred sugar to force the yeast to adapt a highly efficient transport and metabolic pathway for such sugar.

[0053] However, such specialists may not exist in nature, and isolation of such specialists may involve mutagenesis of the yeast strain or strains prior to selection. Mutagenesis of yeast, such as Saccharomyces yeast, preferably Saccharomyces sensu stricto yeast, may be carried out using any method known in the art, including traditional random mutagenesis methods such as radiation and chemical treatment, and recombinant DNA techniques such as site-directed or targeted mutagenesis. Thus, yeast cells may be subjected to random mutagenesis, including UV irradiation, X-ray irradiation, gamma irradiation and treatment with mutagenic agents, or may have been subjected to genetic engineering.

[0054] The term "random mutagenesis" refers to a mutagenesis technique in which the exact site of the mutation is unpredictable and can occur anywhere in the chromosome of a yeast cell or spore. Generally, these methods involve the use of chemicals or radiation to induce at least one mutation.

[0055] "Genetic engineering" is well known to those of skill in the art and refers to the use of biotechnological methods to modify the genome of yeast, whereby modifications in the genomic DNA of the yeast are introduced, preferably at predetermined sites, with predetermined modifications being referred to as site-directed mutagenesis.

[0056] Targeted mutagenesis, also known as site-directed mutagenesis, can use oligonucleotide-guided mutagenesis to generate site-specific mutations in a genomic DNA sequence of interest. Targeted mutagenesis refers to a mutagenesis method that modifies a specific gene in vivo, resulting in changes in gene structure directed to a specific site by programmable RNA-guided nucleases such as TALEN, CRISPR-Cas, zinc finger nucleases, and meganuclease technology.

[0057] Such mutagenesis is preferably carried out by treating the yeast with radiation, such as UV irradiation, X-ray irradiation, gamma irradiation, and / or with a mutagenic agent, preferably a chemical agent, such as NTG (N-methyl-N'-nitro-N-nitrosoguanidine) or EMS (ethyl methanesulfonate). A particularly preferred mutagenesis procedure involves UV irradiation, for example for 10 seconds to 3 minutes, preferably for about 1 to 2 minutes. A preferred method is exposure to UV light (TUV 30 W T8, Philips, Eindhoven, The Netherlands) with an emission peak of 253.7 nm for 0.1 to 10 minutes, preferably 0.5 to 5 minutes, for example for about 90 minutes.

[0058] Such mutations can result in the downregulation or inactivation of one or more genes involved in the uptake and / or metabolism of one or more sugars selected from glucose, maltose, fructose and maltotriose. For example, modification of cell surface glucose sensor Rgt2 and / or Snf3 and downstream nuclear transcription factor Rgt1 results in the repression of genes encoding glucose transporters (Roy et al., 2016. Mol Biol Cell 27:862-871). Thus, mutation of one or more of Rgt2, Snf3 and Rgt1 can result in mutant yeast strains that cannot ferment glucose. Such mutant yeast strains have a preference for sugars selected from maltose and maltotriose.

[0059] Such mutations may result in the downregulation or inactivation of one or more genes involved in the uptake and / or metabolism of one or more sugars selected from glucose, fructose, maltose and maltotriose, as well as the upregulation or activation of genes encoding key enzymes in the uptake, fermentation and / or aerobic degradation of glucose, fructose, maltose and / or maltotriose, such that the mutant yeast cell has a preference for maltotriose, for example, by enhancing the activity of the maltotriose transporter.

[0060] Such mutagenesis, preferably random mutagenesis, is preferably carried out in two or more rounds, each round preferably comprising a mutagenesis step, preferably a mild mutagenesis step, preferably a UV-mediated mutagenesis step, resulting in a moderate survival rate of 20-60%, preferably 40-50%.

[0061] In the first round, the mutant yeasts may be inoculated into a synthetic medium containing one of the sugars glucose, maltose, fructose or maltotriose as the sole carbon source, preferably in a limiting amount of such sugar, This step enriches for mutants that can efficiently consume at least one of glucose, fructose, maltose or maltotriose.

[0062] In a second round, mutant yeasts that have been positively selected on one of the sugars glucose, maltose, fructose or maltotriose, preferably maltose or maltotriose, can be counter-selected by inoculating onto synthetic medium containing the two remaining sugars as the sole carbon source. Yeasts that do not grow or can only grow poorly on the two remaining sugars can be selected for further analysis.

[0063] Optionally, a third round of mutagenesis may include growth on diluted beer wort enriched with one of the sugars glucose, fructose, maltose or maltotriose. Under these conditions, mutants with increased affinity or transport rate for such sugars will be less nutritionally limited and will have a selective advantage compared to other yeasts. For this, the wort may be diluted 2-10 fold, for example 6-fold. Such diluted wort may be enriched with glucose, maltose, fructose or maltotriose, for example 1-20 g L -1 , e.g. 10g L -1 It may be added to increase the relative concentration of glucose, maltose, fructose or maltotriose. To prevent oxygen and nitrogen limitation, ergosterol may be added at a concentration of, for example, 1-100 mg L -1, TWEEN® 80, for example, 100 to 1000 mg L -1 , ammonium sulfate, for example, 1 to 20 mg L -1 May be supplemented.

[0064] Such growth in glucose, maltose, fructose or maltotriose enriched beer wort is preferably carried out by continuous culture. Such continuous culture is carried out for 0.001 to 0.2 h -1 , preferably 0.01 to 0.1 h -1 , for example 0.03 h- 1 At the point where the glucose, fructose, maltose or maltotriose concentration is reduced, single cells are preferably isolated from the culture, for example by FACS sorting. The isolated cells can be plated on synthetic media containing glucose, fructose, maltose or maltotriose as the only carbon source and / or on beer wort enriched with glucose, maltose, fructose or maltotriose, as described herein above, to further select Saccharomyces mutants having a preference for sugars selected from maltose, maltotriose, fructose and glucose.

[0065] If necessary, Saccharomyces mutants having a preference for sugars selected from maltose, maltotriose, fructose and glucose may be counter-selected by growing such mutants on media or plates containing one or more of the remaining sugars. Preferred Saccharomyces mutants may be those that have a preference for a particular sugar selected from maltose, maltotriose, fructose and glucose, but show reduced growth on the other three sugars. Such reduced growth is preferably limited to less than 50% of the unmutated Saccharomyces yeast, less than 25% of the unmutated Saccharomyces yeast, less than 10% of the unmutated Saccharomyces yeast, or most preferably less than 5% of the unmutated Saccharomyces yeast.

[0066] Saccharomyces mutants with a preference for sugars selected from maltose, maltotriose, fructose and glucose can be produced in yeast strains hybridized with a second yeast strain containing additional properties. For example, Saccharomyces cerevisiae mutants can be produced with a preference for fermenting maltose or can be selected without mutation. Such mutants can be hybridized with S. eubayanus strains that are unable to ferment glucose, fructose and / or maltotriose. As a result, interspecies hybrids can be selected that do not or only poorly ferment glucose, fructose and / or maltotriose but have a preference for fermenting maltose.

[0067] Such fermenting yeast strains preferably comprise a mutation resulting in the inactivation of at least one of the putative flavin prenyltransferase (PAD1) and ferulic acid decarboxylase (FDC1) genes, and / or the inactivation of a gene encoding a protein involved in the import of phenolic acids, preferably ferulic acid, or a protein involved in the export of decarboxylated phenolic compounds, preferably 4-vinylguaiacol.

[0068] The fermented beer product so produced is preferably beer, preferably lager beer.

[0069] 4.3 Identification of Saccharomyces yeasts with different sugar consumption characteristics. As known to those skilled in the art, yeast strains having a preference for sugars selected from maltose, maltotriose, fructose and glucose can be produced in several ways. For example, a culture medium, preferably a sterile culture medium, can be produced that contains at least one of the sugars maltose, maltotriose, fructose and glucose. Such a growth medium can be a natural growth medium, a synthetic growth medium, or a combination thereof.

[0070] Preferably, different media are generated, each containing at least one of the sugars maltose, maltotriose, fructose and glucose. For the growth of the particular Saccharomyces yeast strain on each sugar, preferably the initial growth, more preferably the initial exponential growth, is then measured. Such growth rate may be determined by determining the slope of the exponential growth curve, preferably the initial exponential growth curve. Methods for determining such slope are known in the art and may be used to measure the growth curve, preferably the exponential growth curve, according to the formula X=X0*e μτ where X0 is the number of yeast at T=0 and μ is the growth rate, and determining the logarithm of x at time x. Such determined growth rates are preferably the maximum growth rates for growth of the particular Saccharomyces yeast strain on each sugar.

[0071] Alternatively or additionally, a growth medium is provided that contains a combination of sugars, such as wort. Such growth medium preferably contains a specific amount of fermentable sugars, such as 2-500 g / L, 5-200 g / L, including 50 g / L, 75 g / L, 100 g / L, 120 g / L and 150 g / L of maltose, maltotriose, fructose and glucose sugars. After inoculation of such growth medium with a specific Saccharomyces yeast strain, samples are taken from the growth medium at regular intervals, such as every 2 hours, 5 hours, 10 hours, 12 hours or once a day, for example, over a period of 1 day to 2 weeks, including 1 week. Such samples are used to measure, for example, the OD 660 The number of yeast cells can be analyzed by measuring the amount of fermentable sugars and / or their metabolites. Such fermentable sugars preferably include maltose, maltotriose, fructose and glucose, preferably maltose, maltotriose and glucose, more preferably maltose and maltotriose.

[0072] Such preferences are preferably determined by determining the consumption rates of individual sugars, including maltose consumption rate, maltotriose consumption rate, glucose consumption rate and / or fructose consumption rate, which are preferably determined in the exponential phase of the consumption curve of such fermentable sugars, e.g., according to the formula Y=Y0*e (k*X) is determined by fitting an exponential curve of 0.1 to the time points of the exponential growth phase of each individual fermentable sugar consumption curve.

[0073] Such yeast strains preferably have a preference for maltose or maltotriose. Such preference can be expressed as a dimensionless value of the maltose / maltotriose (M2 / M3) kinetic ratio. Examples of M2 / M3 kinetic ratios for individual yeast strains are shown in Table 1 and Figure 2. The M2 / M3 kinetic ratio values ​​ranged from 0.28 to a maximum of 10 for all samples. The maltose specialist S. pastorianus CBS 1483 had an M2 / M3 kinetic ratio in the range of 4-10. The maltotriose specialist Saccharomyces pastorianus CBS 1513 (also known as NCYC396) showed an M2 / M3 kinetic ratio of 0.28-0.40. The generalist strain WS34 / 70 had an M2 / M3 kinetic ratio of 1.82-1.96.

[0074] Thus, a yeast strain with maltose preference can transport and metabolize maltose with higher preference, with a maltose / maltotriose rate ratio of 3 or more, preferably 4 to 10. Maltotriose-preferring yeast can transport and metabolize maltotriose with higher preference, with a maltose / maltotriose rate ratio of less than 2, preferably less than 1, such as 0.01 to 1. Generalist yeast have no preference for the conversion of maltose or maltotriose, with a maltose / maltotriose rate ratio of 1 to 4, preferably between 1.6 to 2.2, for example about 2.

[0075] The maltose / maltotriose rate ratio of an individual yeast strain can be determined without inventive techniques. As shown in Table 1, such maltose / maltotriose rate ratios were determined for a total of 139 individual yeast strains.

[0076] 4.4 A method for producing a fermented beverage using a mixture of Saccharomyces yeasts with different specificities for maltose, maltotriose, fructose and / or glucose. Yeasts have been used since ancient times in baking, brewing and distilling, for example in bread production and in the fermentation of beer and wine. The method of the present invention allows for better mixed sugar fermentation kinetics than the application of a single generalist strain, resulting in improved final fermentation degree and ethanol yield.

[0077] The method of the present invention involves providing crushed cereals, preferably barley, in an aqueous solution, preferably water, to release maltose. Following this malting step, the resulting wort is boiled in the presence of hops and fermented after cooling. Once fermentation is complete, the beer is filtered and bottled. Those skilled in the art will recognize that complete fermentation of beer, such as lager beer, can take up to six weeks, depending on temperature, yeast start, etc.

[0078] During the fermentation process, fermentable sugars are converted to flavor compounds such as alcohols, such as ethanol, CO2, and esters, such as isoamyl acetate. As known to those skilled in the art, factors that affect the appearance and taste of the resulting product include, but are not limited to, the temperature and time of roasting the grains, the temperature and time of steeping, germinating, and kilning the grains, the temperature and time of milling and mashing the grains, the filtering of the mash to produce wort, the temperature and time of boiling the wort, the timing and amount of hops added, the particular hops used, the temperature and time of fermentation, the type of yeast, the mechanical filtering of the yeast or the addition of filtering agents to remove the yeast, and finally the carbonation and packaging of the beer. The conditioning step, which may begin after fermentation but before filtration, gives the yeast time from several days to several weeks to absorb the common off-flavors associated with underconditioned or "green" beers, including sulfur, butter, and green apple.

[0079] In the method of the present invention, the fermentation process is carried out at ambient temperature, preferably below 30° C., such as 6-25° C., 6-24° C., 6-23° C., 6-22° C., 6-21° C., 6-20° C., 6-19° C., 6-18° C., 6-17° C., 6-16° C., 6-15° C., 6-14° C., more preferably 8-13° C., including 9° C., 10° C., 11° C., and 12° C. Lager beer fermentation is generally carried out at 7-13° C. This temperature may depend on the particular yeast strain used in the fermentation process, as known to the skilled person.

[0080] The method of the invention preferably uses a combination of yeast strains with different specificities for fermenting maltose, maltotriose, fructose and / or glucose present in the wort to ethanol, preferably including naturally occurring yeasts of Saccharomyces sensu stricto, preferably S. cerevisiae, S. carlsbergensis, S. pastorianus, S. eubayanus yeasts and / or hybrids thereof, or mutants of Saccharomyces sensu stricto, preferably S. cerevisiae, S. carlsbergensis, S. pastorianus, S. eubayanus yeasts and / or hybrids thereof.

[0081] Each of these yeast strains has been selected for an improved preference for fermenting one of maltose, fructose, maltotriose and glucose over the other two sugars. A preferred yeast is a S. pastorianus yeast, preferably a naturally occurring S. pastorianus yeast such as S. pastorianus strain CBS1483, S. pastorianus strain CBS1513, or a combination of S. pastorianus strain CBS1483 and S. pastorianus strain CBS1513.

[0082] Maltotriose specialists are CBS 1513, NCYC 452, NCYC 1269, NCYC 451, NCYC 457, NCYC 204, NCYC 185, NCYC 1239, NCYC 1146, NCYC 1297, NCYC 1073, NCYC 231, NCYC 1305, CBS 6903, NCYC 203, CBS 2443, NCYC 2339, NCYC 1324, CBS 2898, CBS 7240, NCYC 1526, NCYC 1544, NCYC 2359, NCYC 986, NCYC 1295, NCYC 1516, NCYC 1326, NCYC 2398, NCYC 669, and NCYC 2921.

[0083] The maltose specialist can be selected from NCYC 1262, NCYC 227, NCYC 699, NCYC 487, NCYC 2340, NCYC 1296, NCYC 228, NCYC 534, NCYC 2801, NCYC 1365, NCYC 668, NCYC 450, NCYC 1236, NCYC 75, NCYC 112, NCYC 115, NCYC 177, NCYC 223, NCYC 224, NCYC 240, NCYC 340, NCYC 478, NCYC 479, NCYC 510, NCYC 530, NCYC 965, NCYC 969, NCYC 975, NCYC 987, NCYC 989, NCYC 1322, NCYC 1323, NCYC 229, NCYC 230, NCYC 242, NCYC 392, NCYC 397, NCYC 398, NCYC 511, NCYC 584, NCYC 3420, CBS 12357, CBS 7001, CBS 1174, CBS 1177, CBS 1245, CBS 1386, CBS 1488, CBS 1502, CBS 1504, CBS 1548, CBS 1550, CBS 1551, CBS 1552, CBS 1603, CBS 1605, CBS 1606, CBS 1608, CBS 1665, CBS 2165, CBS 2442, CBS 2945, CBS 2954, CBS 2986, CBS 5156, CBS 6017, yHDPN 421, yHKS 210, yHKS 212, yHKS 509, yHRVM 107, NCYC 966, and CBS 1483.

[0084] The yeast combinations were selected from CBS 1513, NCYC 452, NCYC 1269, NCYC 451, NCYC 457, and NCYC 204, which had the lowest M2 / M3 ratios, and NCYC 75, NCYC 112, NCYC 115, NCYC 177, NCYC 223, NCYC 224, NCYC 240, NCYC 340, NCYC 478, NCYC 479, NCYC 510, NCYC 530, NCYC 965, NCYC 969, NCYC 975, NCYC 987, NCYC 989, NCYC 1322, NCYC 1323, NCYC 229 ...478, NCYC 478, NCYC 478, NCYC 230, NCYC 242, NCYC 392, NCYC 397, NCYC 398, NCYC 511, NCYC 584, NCYC 3420, CBS 12357, CBS 7001, CBS 1174, CBS 1177, CBS 1245, CBS 1386, CBS 1488, CBS 1502, CBS 1504, CBS 1548, CBS 1550, CBS 1551, CBS 1552, CBS 1603, CBS 1605, CBS 1606, CBS 1608, CBS 1665, CBS 2165, CBS 2442, CBS 2945, CBS 2954, CBS 2986, CBS 5156, CBS 6017, yHDPN 421, yHKS 210, yHKS 212, yHKS 509, yHRVM 107, NCYC 966, and any one of CBS 1483.

[0085] Such yeast combinations include any one of CBS 1513, NCYC 452, NCYC 1269, NCYC 451, NCYC 457, and NCYC 204, which have the lowest M2 / M3 velocity ratios, and any one of the generalist strains NCYC 984, NCYC 529, NCYC 2347, NCYC 2837, NCYC 670, NCYC 2426, NCYC 531, WS34 / 70, NCYC 454, NCYC 73, NCYC 2337, NCYC 1250, NCYC 1056, NCYC 456, NCYC 985, NCYC 680, NCYC 2338, NCYC 1026, CBS 1484, CBS 5832, NCYC 204 .... 1341, CMB S33, NCYC 399, NCYC 1342, NCYC 400, NCYC 3419, NCYC 1048, NCYC 1116, NCYC 55, NCYC 453, NCYC 1057, NCYC 1047, CBS 8834, NCYC 967, and any one of NCYC 679.

[0086] The yeast combinations with the highest M2 / M3 ratios are NCYC 75, NCYC 112, NCYC 115, NCYC 177, NCYC 223, NCYC 224, NCYC 240, NCYC 340, NCYC 478, NCYC 479, NCYC 510, NCYC 530, NCYC 965, NCYC 969, NCYC 975, NCYC 987, NCYC 989, NCYC 1322, NCYC 1323, NCYC 229, NCYC 230, NCYC 242, NCYC 392, NCYC 397, NCYC 398, NCYC 511, NCYC 584, NCYC 3420, CBS 12357, CBS 7001, CBS 1174, CBS 1177, CBS 1245, CBS 1386, CBS 1488, CBS 1502, CBS 1504, CBS 1548, CBS 1550, CBS 1551, CBS 1552, CBS 1603, CBS 1605, CBS 1606, CBS 1608, CBS 1665, CBS 2165, CBS 2442, CBS 2945, CBS 2954, CBS 2986, CBS 5156, CBS 6017, yHDPN 421, yHKS 210, yHKS 212, yHKS 509, yHRVM 107, NCYC 966, and CBS 1483, and the generalist strains NCYC 984, NCYC 529, NCYC 2347, NCYC 2837, NCYC 670, NCYC 2426, NCYC 531, WS34 / 70, NCYC 454, NCYC 73, NCYC 2337, NCYC 1250, NCYC 1056, NCYC 456, NCYC 985, NCYC 680, NCYC 2338, NCYC 1026, CBS 1484, CBS 5832, NCYC 1341, CMB S33, NCYC 399, NCYC 1342, NCYC 400, NCYC 3419, NCYC 1048, NCYC 1116, NCYC 55, NCYC 453, NCYC 1057, NCYC 1047, CBS 8834, NCYC 967, and any one of NCYC 679.

[0087] Such combinations are preferably selected from the following: CBS 1513 and NCYC 1262, CBS 1513 and NCYC 227, CBS 1513 and NCYC 699, CBS 1513 and NCYC 487, CBS 1513 and NCYC 2340, CBS 1513 and NCYC 1296, CBS 1513 and NCYC 228, CBS 1513 and NCYC 534, CBS 1513 and NCYC 2801, CBS 1513 and NCYC 1365, CBS 1513 and NCYC 668, CBS 1513 and NCYC 450, CBS 1513 and NCYC 1236, CBS 1513 and NCYC 75, CBS 1513 and NCYC 112, CBS 1513 and NCYC 115, CBS 1513 and NCYC 177, CBS 1513 and NCYC 223, CBS 1513 and NCYC 224, CBS 1513 and NCYC 240, CBS 1513 and NCYC 340, CBS 1513 and NCYC 478, CBS 1513 and NCYC 479, CBS 1513 and NCYC 510, CBS 1513 and NCYC 530, CBS 1513 and NCYC 965, CBS 1513 and NCYC 969, CBS 1513 and NCYC 975, CBS 1513 and NCYC 987, CBS 1513 and NCYC 989, CBS 1513 and NCYC 1322, CBS 1513 and NCYC 1323, CBS 1513 and NCYC 229, CBS 1513 and NCYC 230, CBS 1513 and NCYC 242, CBS 1513 and NCYC 392, CBS 1513 and NCYC 397, CBS 1513 and NCYC 398, CBS 1513 and NCYC 511, CBS 1513 and NCYC 584, CBS 1513 and NCYC 3420, CBS 1513 and CBS 12357, CBS 1513 and CBS 7001, CBS 1513 and CBS 1174, CBS 1513 and CBS 1177, CBS 1513 and CBS 1245, CBS 1513 and CBS 1386, CBS 1513 and CBS 1488, CBS 1513 and CBS 1502, CBS 1513 and CBS 1504, CBS 1513 and CBS1548, CBS 1513 and CBS 1550, CBS 1513 and CBS 1551, CBS 1513 and CBS 1552, CBS 1513 and CBS 1603, CBS 1513 and CBS 1605, CBS 1513 and CBS 1606, CBS 1513 and CBS 1608, CBS 1513 and CBS 1665, CBS 1513 and CBS 2165, CBS 1513 and CBS 2442, CBS 1513 and CBS 2945, CBS 1513 and CBS 2954, CBS 1513 and CBS 2986, CBS 1513 and CBS 5156, CBS 1513 and CBS 6017, CBS 1513 and yHDPN 421, CBS 1513 and yHKS 210, CBS 1513 and yHKS 212, CBS 1513 and yHKS 509, CBS 1513 and yHRVM 107, CBS 1513 and NCYC 966, and CBS 1513 and CBS 1483; NCYC 452 and NCYC 1262, NCYC 452 and NCYC 227, NCYC 452 and NCYC 699, NCYC 452 and NCYC 487, NCYC 452 and NCYC 2340, NCYC 452 and NCYC 1296, NCYC 452 and NCYC 228, NCYC 452 and NCYC 534, NCYC 452 and NCYC 2801, NCYC 452 and NCYC 1365, NCYC 452 and NCYC 668, NCYC 452 and NCYC 450, NCYC 452 and NCYC 1236, NCYC 452 and NCYC 75, NCYC 452 and NCYC 112, NCYC 452 and NCYC 115, NCYC 452 and NCYC 177, NCYC 452 and NCYC 223, NCYC 452 and NCYC 224, NCYC 452 and NCYC 240, NCYC 452 and NCYC 340, NCYC 452 and NCYC 478, NCYC 452 and NCYC 479, NCYC 452 and NCYC 510, NCYC 452 and NCYC 530, NCYC 452 and NCYC 965, NCYC 452 and NCYC 969, NCYC 452 and NCYC 975, NCYC 452 and NCYC 987, NCYC 452 and NCYC989, NCYC 452 and NCYC 1322, NCYC 452 and NCYC 1323, NCYC 452 and NCYC 229, NCYC 452 and NCYC 230, NCYC 452 and NCYC 242, NCYC 452 and NCYC 392, NCYC 452 and NCYC 397, NCYC 452 and NCYC 398, NCYC 452 and NCYC 511, NCYC 452 and NCYC 584, NCYC 452 and NCYC 3420, NCYC 452 and CBS 12357, NCYC 452 and CBS 7001, NCYC 452 and CBS 1174, NCYC 452 and CBS 1177, NCYC 452 and CBS 1245, NCYC 452 and CBS 1386, NCYC 452 and CBS 1488, NCYC 452 and CBS 1502, NCYC 452 and CBS 1504, NCYC 452 and CBS 1548, NCYC 452 and CBS 1550, NCYC 452 and CBS 1551, NCYC 452 and CBS 1552, NCYC 452 and CBS 1603, NCYC 452 and CBS 1605, NCYC 452 and CBS 1606, NCYC 452 and CBS 1608, NCYC 452 and CBS 1665, NCYC 452 and CBS 2165, NCYC 452 and CBS 2442, NCYC 452 and CBS 2945, NCYC 452 and CBS 2954, NCYC 452 and CBS 2986, NCYC 452 and CBS 5156, NCYC 452 and CBS 6017, NCYC 452 and yHDPN 421, NCYC 452 and yHKS 210, NCYC 452 and yHKS 212, NCYC 452 and yHKS 509, NCYC 452 and yHRVM 107, NCYC 452 and NCYC 966, and NCYC 452 and CBS 1483; NCYC 1269 and NCYC 1262, NCYC 1269 and NCYC 227, NCYC 1269 and NCYC 699, NCYC 1269 and NCYC 487, NCYC 1269 and NCYC 2340, NCYC 1269 and NCYC 1296, NCYC 1269 and NCYC 228, NCYC 1269 and NCYC 534, NCYC1269 and NCYC 2801, NCYC 1269 and NCYC 1365, NCYC 1269 and NCYC 668, NCYC 1269 and NCYC 450, NCYC 1269 and NCYC 1236, NCYC 1269 and NCYC 75, NCYC 1269 and NCYC 112, NCYC 1269 and NCYC 115, NCYC 1269 and NCYC 177, NCYC 1269 and NCYC 223, NCYC 1269 and NCYC 224, NCYC 1269 and NCYC 240, NCYC 1269 and NCYC 340, NCYC 1269 and NCYC 478, NCYC 1269 and NCYC 479, NCYC 1269 and NCYC 510, NCYC 1269 and NCYC 530, NCYC 1269 and NCYC 965, NCYC 1269 and NCYC 969, NCYC 1269 and NCYC 975, NCYC 1269 and NCYC 987, NCYC 1269 and NCYC 989, NCYC 1269 and NCYC 1322, NCYC 1269 and NCYC 1323, NCYC 1269 and NCYC 229, NCYC 1269 and NCYC 230, NCYC 1269 and NCYC 242, NCYC 1269 and NCYC 392, NCYC 1269 and NCYC 397, NCYC 1269 and NCYC 398, NCYC 1269 and NCYC 511, NCYC 1269 and NCYC 584, NCYC 1269 and NCYC 3420, NCYC 1269 and CBS 12357, NCYC 1269 and CBS 7001, NCYC 1269 and CBS 1174, NCYC 1269 and CBS 1177, NCYC 1269 and CBS 1245, NCYC 1269 and CBS 1386, NCYC 1269 and CBS 1488, NCYC 1269 and CBS 1502, NCYC 1269 and CBS 1504, NCYC 1269 and CBS 1548, NCYC 1269 and CBS 1550, NCYC 1269 and CBS 1551, NCYC 1269 and CBS 1552, NCYC 1269 and CBS 1603, NCYC 1269 and CBS 1605, NCYC 1269 and CBS 1606, NCYC 1269 and CBS1608, NCYC 1269 and CBS 1665, NCYC 1269 and CBS 2165, NCYC 1269 and CBS 2442, NCYC 1269 and CBS 2945, NCYC 1269 and CBS 2954, NCYC 1269 and CBS 2986, NCYC 1269 and CBS 5156, NCYC 1269 and CBS 6017, NCYC 1269 and yHDPN 421, NCYC 1269 and yHKS 210, NCYC 1269 and yHKS 212, NCYC 1269 and yHKS 509, NCYC 1269 and yHRVM 107, NCYC 1269 and NCYC 966, and NCYC 1269 and CBS 1483; NCYC 451 and NCYC 1262, NCYC 451 and NCYC 227, NCYC 451 and NCYC 699, NCYC 451 and NCYC 487, NCYC 451 and NCYC 2340, NCYC 451 and NCYC 1296, NCYC 451 and NCYC 228, NCYC 451 and NCYC 534, NCYC 451 and NCYC 2801, NCYC 451 and NCYC 1365, NCYC 451 and NCYC 668, NCYC 451 and NCYC 450, NCYC 451 and NCYC 1236, NCYC 451 and NCYC 75, NCYC 451 and NCYC 112, NCYC 451 and NCYC 115, NCYC 451 and NCYC 177, NCYC 451 and NCYC 223, NCYC 451 and NCYC 224, NCYC 451 and NCYC 240, NCYC 451 and NCYC 340, NCYC 451 and NCYC 478, NCYC 451 and NCYC 479, NCYC 451 and NCYC 510, NCYC 451 and NCYC 530, NCYC 451 and NCYC 965, NCYC 451 and NCYC 969, NCYC 451 and NCYC 975, NCYC 451 and NCYC 987, NCYC 451 and NCYC 989, NCYC 451 and NCYC 1322, NCYC 451 and NCYC 1323, NCYC 451 and NCYC 229, NCYC 451 and NCYC 230, NCYC 451 and NCYC 242, NCYC 451 and NCYC392, NCYC 451 and NCYC 397, NCYC 451 and NCYC 398, NCYC 451 and NCYC 511, NCYC 451 and NCYC 584, NCYC 451 and NCYC 3420, NCYC 451 and CBS 12357, NCYC 451 and and CBS 7001, NCYC 451 and CBS 1174, NCYC 451 and CBS 1177, NCYC 451 and CBS 1245, NCYC 451 and CBS 1386, NCYC 451 and CBS 1488, NCYC 451 and CBS 1502, NCYC 451 and CBS 1504, NCYC 451 and CBS 1548, NCYC 451 and CBS 1550, NCYC 451 and CBS 1551, NCYC 451 and CBS 1552, NCYC 451 and CBS 1603, NCYC 451 and CBS 1605, NCYC 451 and CBS 1606, NCYC 451 and CBS 1608, NCYC 451 and CBS 1665, NCYC 451 and CBS 2165, NCYC 451 and CBS 2442, NCYC 451 and CBS 2945, NCYC 451 and CBS 2954, NCYC 451 and CBS 2986, NCYC 451 and CBS 5156, NCYC 451 and CBS 6017, NCYC 451 and yHDPN 421, NCYC 451 and yHKS 210, NCYC 451 and yHKS 212, NCYC 451 and yHKS 509, NCYC 451 and yHRVM 107, NCYC 451 and NCYC 966, and NCYC 451 and CBS 1483; NCYC 457 and NCYC 1262, NCYC 457 and NCYC 227, NCYC 457 and NCYC 699, NCYC 457 and NCYC 487, NCYC 457 and NCYC 2340, NCYC 457 and NCYC 1296, NCYC 457 and NCYC 228, NCYC 457 and NCYC 534, NCYC 457 and NCYC 2801, NCYC 457 and NCYC 1365, NCYC 457 and NCYC 668, NCYC 457 and NCYC 450, NCYC 457 and NCYC 1236, NCYC 457 and NCYC 75, NCYC 457 and NCYC 112, NCYC 457 and NCYC 115, NCYC 457 and NCYC 177, NCYC 457 and NCYC 223, NCYC 457 and NCYC 224, NCYC 457 and NCYC 240, NCYC 457 and NCYC 340, NCYC 457 and NCYC 478, NCYC 457 and NCYC 479, NCYC 457 and NCYC 510, NCYC 457 and NCYC 530, NCYC 457 and NCYC 965, NCYC 457 and NCYC 969, NCYC 457 and NCYC 975, NCYC 457 and NCYC 987, NCYC 457 and NCYC 989, NCYC 457 and NCYC 1322, NCYC 457 and NCYC 1323, NCYC 457 and NCYC 229, NCYC 457 and NCYC 230, NCYC 457 and NCYC 242, NCYC 457 and NCYC 392, NCYC 457 and NCYC 397, NCYC 457 and NCYC 398, NCYC 457 and NCYC 511, NCYC 457 and NCYC 584, NCYC 457 and NCYC 3420, NCYC 457 and CBS 12357, NCYC 457 and CBS 7001, NCYC 457 and CBS 1174, NCYC 457 and CBS 1177, NCYC 457 and CBS 1245, NCYC 457 and CBS 1386, NCYC 457 and CBS 1488, NCYC 457 and CBS 1502, NCYC 457 and CBS 1504, NCYC 457 and CBS 1548, NCYC 457 and CBS1550, NCYC 457 and CBS 1551, NCYC 457 and CBS 1552, NCYC 457 and CBS 1603, NCYC 457 and CBS 1605, NCYC 457 and CBS 1606, NCYC 457 and CBS 1608, NCYC 457 and CBS 1665, NCYC 457 and CBS 2165, NCYC 457 and CBS 2442, NCYC 457 and CBS 2945, NCYC 457 and CBS 2954, NCYC 457 and CBS 2986, NCYC 457 and CBS 5156, NCYC 457 and CBS 6017, NCYC 457 and yHDPN 421, NCYC 457 and yHKS 210, NCYC 457 and yHKS 212, NCYC 457 and yHKS 509, NCYC 457 and yHRVM 107, NCYC 457 and NCYC 966, and NCYC 457 and CBS 1483; NCYC 204 and NCYC 1262, NCYC 204 and NCYC 227, NCYC 204 and NCYC 699, NCYC 204 and NCYC 487, NCYC 204 and NCYC 2340, NCYC 204 and NCYC 1296, NCYC 204 and NCYC 228, NCYC 204 and NCYC 534, NCYC 204 and NCYC 2801, NCYC 204 and NCYC 1365, NCYC 204 and NCYC 668, NCYC 204 and NCYC 450, NCYC 204 and NCYC 1236, NCYC 204 and NCYC 75, NCYC 204 and NCYC 112, NCYC 204 and NCYC 115, NCYC 204 and NCYC 177, NCYC 204 and NCYC 223, NCYC 204 and NCYC 224, NCYC 204 and NCYC 240, NCYC 204 and NCYC 340, NCYC 204 and NCYC 478, NCYC 204 and NCYC 479, NCYC 204 and NCYC 510, NCYC 204 and NCYC 530, NCYC 204 and NCYC 965, NCYC 204 and NCYC 969, NCYC 204 and NCYC 975, NCYC 204 and NCYC 987, NCYC 204 and NCYC 989, NCYC 204 and NCYC1322, NCYC 204 and NCYC 1323, NCYC 204 and NCYC 229, NCYC 204 and NCYC 230, NCYC 204 and NCYC 242, NCYC 204 and NCYC 392, NCYC 204 and NCYC 397, NCYC 204 and NCYC 398, NCYC 204 and NCYC 511, NCYC 204 and NCYC 584, NCYC 204 and NCYC 3420, NCYC 204 and CBS 12357, NCYC 204 and CBS 7001, NCYC 204 and CBS 1174, NCYC 204 and CBS 1177, NCYC 204 and CBS 1245, NCYC 204 and CBS 1386, NCYC 204 and CBS 1488, NCYC 204 and CBS 1502, NCYC 204 and CBS 1504, NCYC 204 and CBS 1548, NCYC 204 and CBS 1550, NCYC 204 and CBS 1551, NCYC 204 and CBS 1552, NCYC 204 and CBS 1603, NCYC 204 and CBS 1605, NCYC 204 and CBS 1606, NCYC 204 and CBS 1608, NCYC 204 and CBS 1665, NCYC 204 and CBS 2165, NCYC 204 and CBS 2442, NCYC 204 and CBS 2945, NCYC 204 and CBS 2954, NCYC 204 and CBS 2986, NCYC 204 and CBS 5156, NCYC 204 and CBS 6017, NCYC 204 and yHDPN 421, NCYC 204 and yHKS 210, NCYC 204 and yHKS 212, NCYC 204 and yHKS 509, NCYC 204 and yHRVM 107, NCYC 204 and NCYC 966, and NCYC 204 and CBS 1483; NCYC 185 and NCYC 1262, NCYC 185 and NCYC 227, NCYC 185 and NCYC 699, NCYC 185 and NCYC 487, NCYC 185 and NCYC 2340, NCYC 185 and NCYC 1296, NCYC 185 and NCYC 228, NCYC 185 and NCYC 534, NCYC 185 and NCYC 2801, NCYC185 and NCYC 1365, NCYC 185 and NCYC 668, NCYC 185 and NCYC 450, NCYC 185 and NCYC 1236, NCYC 185 and NCYC 75, NCYC 185 and NCYC 112, NCYC 185 and NCYC 115, NCYC 185 and NCYC 177, NCYC 185 and NCYC 223, NCYC 185 and NCYC 224, NCYC 185 and NCYC 240, NCYC 185 and NCYC 340, NCYC 185 and NCYC 478, NCYC 185 and NCYC 479, NCYC 185 and NCYC 510, NCYC 185 and NCYC 530, NCYC 185 and NCYC 965, NCYC 185 and NCYC 969, NCYC 185 and NCYC 975, NCYC 185 and NCYC 987, NCYC 185 and NCYC 989, NCYC 185 and NCYC 1322, NCYC 185 and NCYC 1323, NCYC 185 and NCYC 229, NCYC 185 and NCYC 230, NCYC 185 and NCYC 242, NCYC 185 and NCYC 392, NCYC 185 and NCYC 397, NCYC 185 and NCYC 398, NCYC 185 and NCYC 511, NCYC 185 and NCYC 584, NCYC 185 and NCYC 3420, NCYC 185 and CBS 12357, NCYC 185 and CBS 7001, NCYC 185 and CBS 1174, NCYC 185 and CBS 1177, NCYC 185 and CBS 1245, NCYC 185 and CBS 1386, NCYC 185 and CBS 1488, NCYC 185 and CBS 1502, NCYC 185 and CBS 1504, NCYC 185 and CBS 1548, NCYC 185 and CBS 1550, NCYC 185 and CBS 1551, NCYC 185 and CBS 1552, NCYC 185 and CBS 1603, NCYC 185 and CBS 1605, NCYC 185 and CBS 1606, NCYC 185 and CBS 1608, NCYC 185 and CBS 1665, NCYC 185 and CBS 2165, NCYC 185 and CBS 2442, NCYC185 and CBS 2945, NCYC 185 and CBS 2954, NCYC 185 and CBS 2986, NCYC 185 and CBS 5156, NCYC 185 and CBS 6017, NCYC 185 and yHDPN 421, NCYC 185 and yHKS 210, NCYC 185 and yHKS 212, NCYC 185 and yHKS 509, NCYC 185 and yHRVM 107, NCYC 185 and NCYC 966, and NCYC 185 and CBS 1483; NCYC 1239 and NCYC 1262, NCYC 1239 and NCYC 227, NCYC 1239 and NCYC 699, NCYC 1239 and NCYC 487, NCYC 1239 and NCYC 2340, NCYC 1239 and NCYC 1296, NCYC 1239 and NCYC 228, NCYC 1239 and NCYC 534, NCYC 1239 and NCYC 2801, NCYC 1239 and NCYC 1365, NCYC 1239 and NCYC 668, NCYC 1239 and NCYC 450, NCYC 1239 and NCYC 1236, NCYC 1239 and NCYC 75, NCYC 1239 and NCYC 112, NCYC 1239 and NCYC 115, NCYC 1239 and NCYC 177, NCYC 1239 and NCYC 223, NCYC 1239 and NCYC 224, NCYC 1239 and NCYC 240, NCYC 1239 and NCYC 340, NCYC 1239 and NCYC 478, NCYC 1239 and NCYC 479, NCYC 1239 and NCYC 510, NCYC 1239 and NCYC 530, NCYC 1239 and NCYC 965, NCYC 1239 and NCYC 969, NCYC 1239 and NCYC 975, NCYC 1239 and NCYC 987, NCYC 1239 and NCYC 989, NCYC 1239 and NCYC 1322, NCYC 1239 and NCYC 1323, NCYC 1239 and NCYC 229, NCYC 1239 and NCYC 230, NCYC 1239 and NCYC 242, NCYC 1239 and NCYC 392, NCYC 1239 and NCYC 397, NCYC 1239 and NCYC 398, NCYC1239 and NCYC 511, NCYC 1239 and NCYC 584, NCYC 1239 and NCYC 3420, NCYC 1239 and CBS 12357, NCYC 1239 and CBS 7001, NCYC 1239 and CBS 1174, NCYC 1239 and CBS 1177, NCYC 1239 and CBS 1245, NCYC 1239 and CBS 1386, NCYC 1239 and CBS 1488, NCYC 1239 and CBS 1502, NCYC 1239 and CBS 1504, NCYC 1239 and CBS 1548, NCYC 1239 and CBS 1550, NCYC 1239 and CBS 1551, NCYC 1239 and CBS 1552, NCYC 1239 and CBS 1603, NCYC 1239 and CBS 1605, NCYC 1239 and CBS 1606, NCYC 1239 and CBS 1608, NCYC 1239 and CBS 1665, NCYC 1239 and CBS 2165, NCYC 1239 and CBS 2442, NCYC 1239 and CBS 2945, NCYC 1239 and CBS 2954, NCYC 1239 and CBS 2986, NCYC 1239 and CBS 5156, NCYC 1239 and CBS 6017, NCYC 1239 and yHDPN 421, NCYC 1239 and yHKS 210, NCYC 1239 and yHKS 212, NCYC 1239 and yHKS 509, NCYC 1239 and yHRVM 107, NCYC 1239 and NCYC 966, and NCYC 1239 and CBS 1483; NCYC 1146 and NCYC 1262, NCYC 1146 and NCYC 227, NCYC 1146 and NCYC 699, NCYC 1146 and NCYC 487, NCYC 1146 and NCYC 2340, NCYC 1146 and NCYC 1296, NCYC 1146 and NCYC 228, NCYC 1146 and NCYC 534, NCYC 1146 and NCYC 2801, NCYC 1146 and NCYC 1365, NCYC 1146 and NCYC 668, NCYC 1146 and NCYC 450, NCYC 1146 and NCYC 1236, NCYC 1146 and NCYC 75, NCYC 1146 and NCYC 112, NCYC 1146 and NCYC 115, NCYC 1146 and NCYC 177, NCYC 1146 and NCYC 223, NCYC 1146 and NCYC 224, NCYC 1146 and NCYC 240, NCYC 1146 and NCYC 340, NCYC1146 and NCYC 478, NCYC 1146 and NCYC 479, NCYC 1146 and NCYC 510, NCYC 1146 and NCYC 530, NCYC 1146 and NCYC 965, NCYC 1146 and NCYC 969, NCYC 1146 and NCYC 975, NCYC 1146 and NCYC 987, NCYC 1146 and NCYC 989, NCYC 1146 and NCYC 1322, NCYC 1146 and NCYC 1323, NCYC 1146 and NCYC 229, NCYC 1146 and NCYC 230, NCYC 1146 and NCYC 242, NCYC 1146 and NCYC 392, NCYC 1146 and NCYC 397, NCYC 1146 and NCYC 398, NCYC 1146 and NCYC 511, NCYC 1146 and NCYC 584, NCYC 1146 and NCYC 3420, NCYC 1146 and CBS 12357, NCYC 1146 and CBS 7001, NCYC 1146 and CBS 1174, NCYC 1146 and CBS 1177, NCYC 1146 and CBS 1245, NCYC 1146 and CBS 1386, NCYC 1146 and CBS 1488, NCYC 1146 and CBS 1502, NCYC 1146 and CBS 1504, NCYC 1146 and CBS 1548, NCYC 1146 and CBS 1550, NCYC 1146 and CBS 1551, NCYC 1146 and CBS 1552, NCYC 1146 and CBS 1603, NCYC 1146 and CBS 1605, NCYC 1146 and CBS 1606, NCYC 1146 and CBS 1608, NCYC 1146 and CBS 1665, NCYC 1146 and CBS 2165, NCYC 1146 and CBS 2442, NCYC 1146 and CBS 2945, NCYC 1146 and CBS 2954, NCYC 1146 and CBS 2986, NCYC 1146 and CBS 5156, NCYC 1146 and CBS 6017, NCYC 1146 and yHDPN 421, NCYC 1146 and yHKS 210, NCYC 1146 and yHKS 212, NCYC 1146 and yHKS 509, NCYC 1146 and yHRVM107, NCYC 1146 and NCYC 966, and NCYC 1146 and CBS 1483; NCYC 1297 and NCYC 1262, NCYC 1297 and NCYC 227, NCYC 1297 and NCYC 699, NCYC 1297 and NCYC 487, NCYC 1297 and NCYC 2340, NCYC 1297 and NCYC 1296, NCYC 1297 and NCYC 228, NCYC 1297 and NCYC 534, NCYC 1297 and NCYC 2801, NCYC 1297 and NCYC 1365, NCYC 1297 and NCYC 668, NCYC 1297 and NCYC 450, NCYC 1297 and NCYC 1236, NCYC 1297 and NCYC 75, NCYC 1297 and NCYC 112, NCYC 1297 and NCYC 115, NCYC 1297 and NCYC 177, NCYC 1297 and NCYC 223, NCYC 1297 and NCYC 224, NCYC 1297 and NCYC 240, NCYC 1297 and NCYC 340, NCYC 1297 and NCYC 478, NCYC 1297 and NCYC 479, NCYC 1297 and NCYC 510, NCYC 1297 and NCYC 530, NCYC 1297 and NCYC 965, NCYC 1297 and NCYC 969, NCYC 1297 and NCYC 975, NCYC 1297 and NCYC 987, NCYC 1297 and NCYC 989, NCYC 1297 and NCYC 1322, NCYC 1297 and NCYC 1323, NCYC 1297 and NCYC 229, NCYC 1297 and NCYC 230, NCYC 1297 and NCYC 242, NCYC 1297 and NCYC 392, NCYC 1297 and NCYC 397, NCYC 1297 and NCYC 398, NCYC 1297 and NCYC 511, NCYC 1297 and NCYC 584, NCYC 1297 and NCYC 3420, NCYC 1297 and CBS 12357, NCYC 1297 and CBS 7001, NCYC 1297 and CBS 1174, NCYC 1297 and CBS 1177, NCYC 1297 and CBS 1245, NCYC 1297 and CBS1386, NCYC 1297 and CBS 1488, NCYC 1297 and CBS 1502, NCYC 1297 and CBS 1504, NCYC 1297 and CBS 1548, NCYC 1297 and CBS 1550, NCYC 1297 and CBS 1551, NCYC 1297 and CBS 1552, NCYC 1297 and CBS 1603, NCYC 1297 and CBS 1605, NCYC 1297 and CBS 1606, NCYC 1297 and CBS 1608, NCYC 1297 and CBS 1665, NCYC 1297 and CBS 2165, NCYC 1297 and CBS 2442, NCYC 1297 and CBS 2945, NCYC 1297 and CBS 2954, NCYC 1297 and CBS 2986, NCYC 1297 and CBS 5156, NCYC 1297 and CBS 6017, NCYC 1297 and yHDPN 421, NCYC 1297 and yHKS 210, NCYC 1297 and yHKS 212, NCYC 1297 and yHKS 509, NCYC 1297 and yHRVM 107, NCYC 1297 and NCYC 966, and NCYC 1297 and CBS 1483; NCYC 1073 and NCYC 1262, NCYC 1073 and NCYC 227, NCYC 1073 and NCYC 699, NCYC 1073 and NCYC 487, NCYC 1073 and NCYC 2340, NCYC 1073 and NCYC 1296, NCYC 1073 and NCYC 228, NCYC 1073 and NCYC 534, NCYC 1073 and NCYC 2801, NCYC 1073 and NCYC 1365, NCYC 1073 and NCYC 668, NCYC 1073 and NCYC 450, NCYC 1073 and NCYC 1236, NCYC 1073 and NCYC 75, NCYC 1073 and NCYC 112, NCYC 1073 and NCYC 115, NCYC 1073 and NCYC 177, NCYC 1073 and NCYC 223, NCYC 1073 and NCYC 224, NCYC 1073 and NCYC 240, NCYC 1073 and NCYC 340, NCYC 1073 and NCYC 478, NCYC 1073 and NCYC 479, NCYC1073 and NCYC 510, NCYC 1073 and NCYC 530, NCYC 1073 and NCYC 965, NCYC 1073 and NCYC 969, NCYC 1073 and NCYC 975, NCYC 1073 and NCYC 987, NCYC 1073 and NCYC 989, NCYC 1073 and NCYC 1322, NCYC 1073 and NCYC 1323, NCYC 1073 and NCYC 229, NCYC 1073 and NCYC 230, NCYC 1073 and NCYC 242, NCYC 1073 and NCYC 392, NCYC 1073 and NCYC 397, NCYC 1073 and NCYC 398, NCYC 1073 and NCYC 511, NCYC 1073 and NCYC 584, NCYC 1073 and NCYC 3420, NCYC 1073 and CBS 12357, NCYC 1073 and CBS 7001, NCYC 1073 and CBS 1174, NCYC 1073 and CBS 1177, NCYC 1073 and CBS 1245, NCYC 1073 and CBS 1386, NCYC 1073 and CBS 1488, NCYC 1073 and CBS 1502, NCYC 1073 and CBS 1504, NCYC 1073 and CBS 1548, NCYC 1073 and CBS 1550, NCYC 1073 and CBS 1551, NCYC 1073 and CBS 1552, NCYC 1073 and CBS 1603, NCYC 1073 and CBS 1605, NCYC 1073 and CBS 1606, NCYC 1073 and CBS 1608, NCYC 1073 and CBS 1665, NCYC 1073 and CBS 2165, NCYC 1073 and CBS 2442, NCYC 1073 and CBS 2945, NCYC 1073 and CBS 2954, NCYC 1073 and CBS 2986, NCYC 1073 and CBS 5156, NCYC 1073 and CBS 6017, NCYC 1073 and yHDPN 421, NCYC 1073 and yHKS 210, NCYC 1073 and yHKS 212, NCYC 1073 and yHKS 509, NCYC 1073 and yHRVM 107, NCYC 1073 and NCYC 966, and NCYC 1073 and CBS1483; NCYC 231 and NCYC 1262, NCYC 231 and NCYC 227, NCYC 231 and NCYC 699, NCYC 231 and NCYC 487, NCYC 231 and NCYC 2340, NCYC 231 and NCYC 1296, NCYC 231 and NCYC 228 , NCYC 231 and NCYC 534, NCYC 231 and NCYC 2801, NCYC 231 and NCYC 1365, NCYC 231 and NCYC 668, NCYC 231 and NCYC 450, NCYC 231 and NCYC 1236, NCYC 231 and NCYC 75, NCYC 231 and NCYC 112, NCYC 231 and NCYC 115, NCYC 231 and NCYC 177, NCYC 231 and NCYC 223, NCYC 231 and NCYC 224, NCYC 231 and NCYC 240, NCYC 231 and NCYC 340, NCYC 231 and NCYC 478, NCYC 231 and NCYC 479, NCYC 231 and NCYC 510, NCYC 231 and NCYC 530, NCYC 231 and NCYC 965, NCYC 231 and NCYC 969, NCYC 231 and NCYC 975, NCYC 231 and NCYC 987, NCYC 231 and NCYC 989, NCYC 231 and NCYC 1322, NCYC 231 and NCYC 1323, NCYC 231 and NCYC 229, NCYC 231 and NCYC 230, NCYC 231 and NCYC 242, NCYC 231 and NCYC 392, NCYC 231 and NCYC 397, NCYC 231 and NCYC 398, NCYC 231 and NCYC 511, NCYC 231 and NCYC 584, NCYC 231 and NCYC 3420, NCYC 231 and CBS 12357, NCYC 231 and CBS 7001, NCYC 231 and CBS 1174, NCYC 231 and CBS 1177, NCYC 231 and CBS 1245, NCYC 231 and CBS 1386, NCYC 231 and CBS 1488, NCYC 231 and CBS 1502, NCYC 231 and CBS 1504, NCYC 231 and CBS 1548, NCYC 231 and CBS 1550, NCYC 231 and CBS 1551, NCYC 231 and CBS 1552, NCYC 231 and CBS 1603, NCYC 231 and CBS 1605, NCYC 231 and CBS 1606, NCYC 231 and CBS 1608, NCYC 231 and CBS 1665, NCYC231 and CBS 2165, NCYC 231 and CBS 2442, NCYC 231 and CBS 2945, NCYC 231 and CBS 2954, NCYC 231 and CBS 2986, NCYC 231 and CBS 5156, NCYC 231 and CBS 6017, NCYC 231 and yHDPN 421, NCYC 231 and yHKS 210, NCYC 231 and yHKS 212, NCYC 231 and yHKS 509, NCYC 231 and yHRVM 107, NCYC 231 and NCYC 966, and NCYC 231 and CBS 1483; NCYC 1305 and NCYC 1262, NCYC 1305 and NCYC 227, NCYC 1305 and NCYC 699, NCYC 1305 and NCYC 487, NCYC 1305 and NCYC 2340, NCYC 1305 and NCYC 1296, NCYC 1305 and NCYC 228, NCYC 1305 and NCYC 534, NCYC 1305 and NCYC 2801, NCYC 1305 and NCYC 1365, NCYC 1305 and NCYC 668, NCYC 1305 and NCYC 450, NCYC 1305 and NCYC 1236, NCYC 1305 and NCYC 75, NCYC 1305 and NCYC 112, NCYC 1305 and NCYC 115, NCYC 1305 and NCYC 177, NCYC 1305 and NCYC 223, NCYC 1305 and NCYC 224, NCYC 1305 and NCYC 240, NCYC 1305 and NCYC 340, NCYC 1305 and NCYC 478, NCYC 1305 and NCYC 479, NCYC 1305 and NCYC 510, NCYC 1305 and NCYC 530, NCYC 1305 and NCYC 965, NCYC 1305 and NCYC 969, NCYC 1305 and NCYC 975, NCYC 1305 and NCYC 987, NCYC 1305 and NCYC 989, NCYC 1305 and NCYC 1322, NCYC 1305 and NCYC 1323, NCYC 1305 and NCYC 229, NCYC 1305 and NCYC 230, NCYC 1305 and NCYC 242, NCYC 1305 and NCYC 392, NCYC1305 and NCYC 397, NCYC 1305 and NCYC 398, NCYC 1305 and NCYC 511, NCYC 1305 and NCYC 584, NCYC 1305 and NCYC 3420, NCYC 1305 and CBS 12357, NCYC 1305 and CBS 7001, NCYC 1305 and CBS 1174, NCYC 1305 and CBS 1177, NCYC 1305 and CBS 1245, NCYC 1305 and CBS 1386, NCYC 1305 and CBS 1488, NCYC 1305 and CBS 1502, NCYC 1305 and CBS 1504, NCYC 1305 and CBS 1548, NCYC 1305 and CBS 1550, NCYC 1305 and CBS 1551, NCYC 1305 and CBS 1552, NCYC 1305 and CBS 1603, NCYC 1305 and CBS 1605, NCYC 1305 and CBS 1606, NCYC 1305 and CBS 1608, NCYC 1305 and CBS 1665, NCYC 1305 and CBS 2165, NCYC 1305 and CBS 2442, NCYC 1305 and CBS 2945, NCYC 1305 and CBS 2954, NCYC 1305 and CBS 2986, NCYC 1305 and CBS 5156, NCYC 1305 and CBS 6017, NCYC 1305 and yHDPN 421, NCYC 1305 and yHKS 210, NCYC 1305 and yHKS 212, NCYC 1305 and yHKS 509, NCYC 1305 and yHRVM 107, NCYC 1305 and NCYC 966, and NCYC 1305 and CBS 1483; CBS 6903 and NCYC 1262, CBS 6903 and NCYC 227, CBS 6903 and NCYC 699, CBS 6903 and NCYC 487, CBS 6903 and NCYC 2340, CBS 6903 and NCYC 1296, CBS 6903 and NCYC 228, CBS 6903 and NCYC 534, CBS 6903 and NCYC 2801, CBS 6903 and NCYC 1365, CBS 6903 and NCYC 668, CBS 6903 and NCYC 450, CBS 6903 and NCYC 1236, CBS6903 and NCYC 75, CBS 6903 and NCYC 112, CBS 6903 and NCYC 115, CBS 6903 and NCYC 177, CBS 6903 and NCYC 223, CBS 6903 and NCYC 224, CBS 6903 and NCYC 240, CBS 6903 and NCYC 340, CBS 6903 and NCYC 478, CBS 6903 and NCYC 479, CBS 6903 and NCYC 510, CBS 6903 and NCYC 530, CBS 6903 and NCYC 965, CBS 6903 and NCYC 969, CBS 6903 and NCYC 975, CBS 6903 and NCYC 987, CBS 6903 and NCYC 989, CBS 6903 and NCYC 1322, CBS 6903 and NCYC 1323, CBS 6903 and NCYC 229, CBS 6903 and NCYC 230, CBS 6903 and NCYC 242, CBS 6903 and NCYC 392, CBS 6903 and NCYC 397, CBS 6903 and NCYC 398, CBS 6903 and NCYC 511, CBS 6903 and NCYC 584, CBS 6903 and NCYC 3420, CBS 6903 and CBS 12357, CBS 6903 and CBS 7001, CBS 6903 and CBS 1174, CBS 6903 and CBS 1177, CBS 6903 and CBS 1245, CBS 6903 and CBS 1386, CBS 6903 and CBS 1488, CBS 6903 and CBS 1502, CBS 6903 and CBS 1504, CBS 6903 and CBS 1548, CBS 6903 and CBS 1550, CBS 6903 and CBS 1551, CBS 6903 and CBS 1552, CBS 6903 and CBS 1603, CBS 6903 and CBS 1605, CBS 6903 and CBS 1606, CBS 6903 and CBS 1608, CBS 6903 and CBS 1665, CBS 6903 and CBS 2165, CBS 6903 and CBS 2442, CBS 6903 and CBS 2945, CBS 6903 and CBS 2954, CBS 6903 and CBS 2986, CBS 6903 and CBS 5156, CBS 6903 and CBS6017, CBS 6903 and yHDPN 421, CBS 6903 and yHKS 210, CBS 6903 and yHKS 212, CBS 6903 and yHKS 509, CBS 6903 and yHRVM 107, CBS 6903 and NCYC 966, and CBS 6903 and CBS 1483; NCYC 203 and NCYC 1262, NCYC 203 and NCYC 227, NCYC 203 and NCYC 699, NCYC 203 and NCYC 487, NCYC 203 and NCYC 2340, NCYC 203 and NCYC 1296, NCYC 203 and NCYC 228, NCYC 203 and NCYC 534, NCYC 203 and NCYC 2801, NCYC 203 and NCYC 1365, NCYC 203 and NCYC 668, NCYC 203 and NCYC 450, NCYC 203 and NCYC 1236, NCYC 203 and NCYC 75, NCYC 203 and NCYC 112, NCYC 203 and NCYC 115, NCYC 203 and NCYC 177, NCYC 203 and NCYC 223, NCYC 203 and NCYC 224, NCYC 203 and NCYC 240, NCYC 203 and NCYC 340, NCYC 203 and NCYC 478, NCYC 203 and NCYC 479, NCYC 203 and NCYC 510, NCYC 203 and NCYC 530, NCYC 203 and NCYC 965, NCYC 203 and NCYC 969, NCYC 203 and NCYC 975, NCYC 203 and NCYC 987, NCYC 203 and NCYC 989, NCYC 203 and NCYC 1322, NCYC 203 and NCYC 1323, NCYC 203 and NCYC 229, NCYC 203 and NCYC 230, NCYC 203 and NCYC 242, NCYC 203 and NCYC 392, NCYC 203 and NCYC 397, NCYC 203 and NCYC 398, NCYC 203 and NCYC 511, NCYC 203 and NCYC 584, NCYC 203 and NCYC 3420, NCYC 203 and CBS 12357, NCYC 203 and CBS 7001, NCYC 203 and CBS 1174, NCYC203 and CBS 1177, NCYC 203 and CBS 1245, NCYC 203 and CBS 1386, NCYC 203 and CBS 1488, NCYC 203 and CBS 1502, NCYC 203 and CBS 1504, NCYC 203 and CBS 1548 , NCYC 203 and CBS 1550, NCYC 203 and CBS 1551, NCYC 203 and CBS 1552, NCYC 203 and CBS 1603, NCYC 203 and CBS 1605, NCYC 203 and CBS 1606, NCYC 203 and CBS 1608, NCYC 203 and CBS 1665, NCYC 203 and CBS 2165, NCYC 203 and CBS 2442, NCYC 203 and CBS 2945, NCYC 203 and CBS 2954, NCYC 203 and CBS 2986, NCYC 203 and CBS 5156, NCYC 203 and CBS 6017, NCYC 203 and yHDPN 421, NCYC 203 and yHKS 210, NCYC 203 and yHKS 212, NCYC 203 and yHKS 509, NCYC 203 and yHRVM 107, NCYC 203 and NCYC 966, and NCYC 203 and CBS 1483; CBS 2443 and NCYC 1262, CBS 2443 and NCYC 227, CBS 2443 and NCYC 699, CBS 2443 and NCYC 487, CBS 2443 and NCYC 2340, CBS 2443 and NCYC 1296, CBS 2443 and NCYC 228, CBS 2443 and NCYC 534, CBS 2443 and NCYC 2801, CBS 2443 and NCYC 1365, CBS 2443 and NCYC 668, CBS 2443 and NCYC 450, CBS 2443 and NCYC 1236, CBS 2443 and NCYC 75, CBS 2443 and NCYC 112, CBS 2443 and NCYC 115, CBS 2443 and NCYC 177, CBS 2443 and NCYC 223, CBS 2443 and NCYC 224, CBS 2443 and NCYC 240, CBS 2443 and NCYC 340, CBS 2443 and NCYC 478, CBS 2443 and NCYC 479, CBS 2443 and NCYC 510, CBS 2443 and NCYC 530, CBS 2443 and NCYC 965, CBS 2443 and NCYC 969, CBS 2443 and NCYC 975, CBS 2443 and NCYC 987, CBS 2443 and NCYC989, CBS 2443 and NCYC 1322, CBS 2443 and NCYC 1323, CBS 2443 and NCYC 229, CBS 2443 and NCYC 230, CBS 2443 and NCYC 242, CBS 2443 and NCYC 392, CBS 2443 and NCYC 397, CBS 2443 and NCYC 398, CBS 2443 and NCYC 511, CBS 2443 and NCYC 584, CBS 2443 and NCYC 3420, CBS 2443 and CBS 12357, CBS 2443 and CBS 7001, CBS 2443 and CBS 1174, CBS 2443 and CBS 1177, CBS 2443 and CBS 1245, CBS 2443 and CBS 1386, CBS 2443 and CBS 1488, CBS 2443 and CBS 1502, CBS 2443 and CBS 1504, CBS 2443 and CBS 1548, CBS 2443 and CBS 1550, CBS 2443 and CBS 1551, CBS 2443 and CBS 1552, CBS 2443 and CBS 1603, CBS 2443 and CBS 1605, CBS 2443 and CBS 1606, CBS 2443 and CBS 1608, CBS 2443 and CBS 1665, CBS 2443 and CBS 2165, CBS 2443 and CBS 2442, CBS 2443 and CBS 2945, CBS 2443 and CBS 2954, CBS 2443 and CBS 2986, CBS 2443 and CBS 5156, CBS 2443 and CBS 6017, CBS 2443 and yHDPN 421, CBS 2443 and yHKS 210, CBS 2443 and yHKS 212, CBS 2443 and yHKS 509, CBS 2443 and yHRVM 107, CBS 2443 and NCYC 966, and CBS 2443 and CBS 1483; NCYC 2339 and NCYC 1262, NCYC 2339 and NCYC 227, NCYC 2339 and NCYC 699, NCYC 2339 and NCYC 487, NCYC 2339 and NCYC 2340, NCYC 2339 and NCYC 1296, NCYC 2339 and NCYC 228, NCYC 2339 and NCYC 534, NCYC2339 and NCYC 2801, NCYC 2339 and NCYC 1365, NCYC 2339 and NCYC 668, NCYC 2339 and NCYC 450, NCYC 2339 and NCYC 1236, NCYC 2339 and NCYC 75, NCYC 2339 and NCYC 112, NCYC 2339 and NCYC 115, NCYC 2339 and NCYC 177, NCYC 2339 and NCYC 223, NCYC 2339 and NCYC 224, NCYC 2339 and NCYC 240, NCYC 2339 and NCYC 340, NCYC 2339 and NCYC 478, NCYC 2339 and NCYC 479, NCYC 2339 and NCYC 510, NCYC 2339 and NCYC 530, NCYC 2339 and NCYC 965, NCYC 2339 and NCYC 969, NCYC 2339 and NCYC 975, NCYC 2339 and NCYC 987, NCYC 2339 and NCYC 989, NCYC 2339 and NCYC 1322, NCYC 2339 and NCYC 1323, NCYC 2339 and NCYC 229, NCYC 2339 and NCYC 230, NCYC 2339 and NCYC 242, NCYC 2339 and NCYC 392, NCYC 2339 and NCYC 397, NCYC 2339 and NCYC 398, NCYC 2339 and NCYC 511, NCYC 2339 and NCYC 584, NCYC 2339 and NCYC 3420, NCYC 2339 and CBS 12357, NCYC 2339 and CBS 7001, NCYC 2339 and CBS 1174, NCYC 2339 and CBS 1177, NCYC 2339 and CBS 1245, NCYC 2339 and CBS 1386, NCYC 2339 and CBS 1488, NCYC 2339 and CBS 1502, NCYC 2339 and CBS 1504, NCYC 2339 and CBS 1548, NCYC 2339 and CBS 1550, NCYC 2339 and CBS 1551, NCYC 2339 and CBS 1552, NCYC 2339 and CBS 1603, NCYC 2339 and CBS 1605, NCYC 2339 and CBS 1606, NCYC 2339 and CBS1608, NCYC 2339 and CBS 1665, NCYC 2339 and CBS 2165, NCYC 2339 and CBS 2442, NCYC 2339 and CBS 2945, NCYC 2339 and CBS 2954, NCYC 2339 and CBS 2986, NCYC 2339 and CBS 5156, NCYC 2339 and CBS 6017, NCYC 2339 and yHDPN 421, NCYC 2339 and yHKS 210, NCYC 2339 and yHKS 212, NCYC 2339 and yHKS 509, NCYC 2339 and yHRVM 107, NCYC 2339 and NCYC 966, and NCYC 2339 and CBS 1483; NCYC 1324 and NCYC 1262, NCYC 1324 and NCYC 227, NCYC 1324 and NCYC 699, NCYC 1324 and NCYC 487, NCYC 1324 and NCYC 2340, NCYC 1324 and NCYC 1296, NCYC 1324 and NCYC 228, NCYC 1324 and NCYC 534, NCYC 1324 and NCYC 2801, NCYC 1324 and NCYC 1365, NCYC 1324 and NCYC 668, NCYC 1324 and NCYC 450, NCYC 1324 and NCYC 1236, NCYC 1324 and NCYC 75, NCYC 1324 and NCYC 112, NCYC 1324 and NCYC 115, NCYC 1324 and NCYC 177, NCYC 1324 and NCYC 223, NCYC 1324 and NCYC 224, NCYC 1324 and NCYC 240, NCYC 1324 and NCYC 340, NCYC 1324 and NCYC 478, NCYC 1324 and NCYC 479, NCYC 1324 and NCYC 510, NCYC 1324 and NCYC 530, NCYC 1324 and NCYC 965, NCYC 1324 and NCYC 969, NCYC 1324 and NCYC 975, NCYC 1324 and NCYC 987, NCYC 1324 and NCYC 989, NCYC 1324 and NCYC 1322, NCYC 1324 and NCYC 1323, NCYC 1324 and NCYC 229, NCYC 1324 and NCYC230, NCYC 1324 and NCYC 242, NCYC 1324 and NCYC 392, NCYC 1324 and NCYC 397, NCYC 1324 and NCYC 398, NCYC 1324 and NCYC 511, NCYC 1324 and NCYC 584, NCYC 1324 and NCYC 3420, NCYC 1324 and CBS 12357, NCYC 1324 and CBS 7001, NCYC 1324 and CBS 1174, NCYC 1324 and CBS 1177, NCYC 1324 and CBS 1245, NCYC 1324 and CBS 1386, NCYC 1324 and CBS 1488, NCYC 1324 and CBS 1502, NCYC 1324 and CBS 1504, NCYC 1324 and CBS 1548, NCYC 1324 and CBS 1550, NCYC 1324 and CBS 1551, NCYC 1324 and CBS 1552, NCYC 1324 and CBS 1603, NCYC 1324 and CBS 1605, NCYC 1324 and CBS 1606, NCYC 1324 and CBS 1608, NCYC 1324 and CBS 1665, NCYC 1324 and CBS 2165, NCYC 1324 and CBS 2442, NCYC 1324 and CBS 2945, NCYC 1324 and CBS 2954, NCYC 1324 and CBS 2986, NCYC 1324 and CBS 5156, NCYC 1324 and CBS 6017, NCYC 1324 and yHDPN 421, NCYC 1324 and yHKS 210, NCYC 1324 and yHKS 212, NCYC 1324 and yHKS 509, NCYC 1324 and yHRVM 107, NCYC 1324 and NCYC 966, and NCYC 1324 and CBS 1483; CBS 2898 and NCYC 1262, CBS 2898 and NCYC 227, CBS 2898 and NCYC 699, CBS 2898 and NCYC 487, CBS 2898 and NCYC 2340, CBS 2898 and NCYC 1296, CBS 2898 and NCYC 228, CBS 2898 and NCYC 534, CBS 2898 and NCYC 2801, CBS 2898 and NCYC 1365, CBS2898 and NCYC 668, CBS 2898 and NCYC 450, CBS 2898 and NCYC 1236, CBS 2898 and NCYC 75, CBS 2898 and NCYC 112, CBS 2898 and NCYC 115, CBS 2898 and NCYC 177, CBS 2898 and NCYC 2 23, CBS 2898 and NCYC 224, CBS 2898 and NCYC 240, CBS 2898 and NCYC 340, CBS 2898 and NCYC 478, CBS 2898 and NCYC 479, CBS 2898 and NCYC 510, CBS 2898 and NCYC 530, CBS 2898 and NCYC 965, CBS 2898 and NCYC 969, CBS 2898 and NCYC 975, CBS 2898 and NCYC 987, CBS 2898 and NCYC 989, CBS 2898 and NCYC 1322, CBS 2898 and NCYC 1323, CBS 2898 and NCYC 229, CBS 2898 and NCYC 230, CBS 2898 and NCYC 242, CBS 2898 and NCYC 392, CBS 2898 and NCYC 397, CBS 2898 and NCYC 398, CBS 2898 and NCYC 511, CBS 2898 and NCYC 584, CBS 2898 and NCYC 3420, CBS 2898 and CBS 12357, CBS 2898 and CBS 7001, CBS 2898 and CBS 1174, CBS 2898 and CBS 1177, CBS 2898 and CBS 1245, CBS 2898 and CBS 1386, CBS 2898 and CBS 1488, CBS 2898 and CBS 1502, CBS 2898 and CBS 1504, CBS 2898 and CBS 1548, CBS 2898 and CBS 1550, CBS 2898 and CBS 1551, CBS 2898 and CBS 1552, CBS 2898 and CBS 1603, CBS 2898 and CBS 1605, CBS 2898 and CBS 1606, CBS 2898 and CBS 1608, CBS 2898 and CBS 1665, CBS 2898 and CBS 2165, CBS 2898 and CBS 2442, CBS 2898 and CBS 2945, CBS 2898 and CBS 2954, CBS 2898 and CBS 2986, CBS 2898 and CBS 5156, CBS 2898 and CBS 6017, CBS 2898 and yHDPN 421, CBS 2898 and yHKS 210, CBS 2898 and yHKS 212, CBS 2898 and yHKS 509, CBS2898 and yHRVM 107, CBS 2898 and NCYC 966, and CBS 2898 and CBS 1483; CBS 7240 and NCYC 1262, CBS 7240 and NCYC 227, CBS 7240 and NCYC 699, CBS 7240 and NCYC 487, CBS 7240 and NCYC 2340, CBS 7240 and NCYC 1296, CBS 7240 and NCYC 228, CBS 7240 and NCYC 534, CBS 7240 and NCYC 2801, CBS 7240 and NCYC 1365, CBS 7240 and NCYC 668, CBS 7240 and NCYC 450, CBS 7240 and NCYC 1236, CBS 7240 and NCYC 75, CBS 7240 and NCYC 112, CBS 7240 and NCYC 115, CBS 7240 and NCYC 177, CBS 7240 and NCYC 223, CBS 7240 and NCYC 224, CBS 7240 and NCYC 240, CBS 7240 and NCYC 340, CBS 7240 and NCYC 478, CBS 7240 and NCYC 479, CBS 7240 and NCYC 510, CBS 7240 and NCYC 530, CBS 7240 and NCYC 965, CBS 7240 and NCYC 969, CBS 7240 and NCYC 975, CBS 7240 and NCYC 987, CBS 7240 and NCYC 989, CBS 7240 and NCYC 1322, CBS 7240 and NCYC 1323, CBS 7240 and NCYC 229, CBS 7240 and NCYC 230, CBS 7240 and NCYC 242, CBS 7240 and NCYC 392, CBS 7240 and NCYC 397, CBS 7240 and NCYC 398, CBS 7240 and NCYC 511, CBS 7240 and NCYC 584, CBS 7240 and NCYC 3420, CBS 7240 and CBS 12357, CBS 7240 and CBS 7001, CBS 7240 and CBS 1174, CBS 7240 and CBS 1177, CBS 7240 and CBS 1245, CBS 7240 and CBS 1386, CBS 7240 and CBS 1488, CBS 7240 and CBS1502, CBS 7240 and CBS 1504, CBS 7240 and CBS 1548, CBS 7240 and CBS 1550, CBS 7240 and CBS 1551, CBS 7240 and CBS 1552, CBS 7240 and CBS 1603, CBS 7240 and CBS 1605, CBS 7240 and CBS 1606, CBS 7240 and CBS 1608, CBS 7240 and CBS 1665, CBS 7240 and CBS 2165, CBS 7240 and CBS 2442, CBS 7240 and CBS 2945, CBS 7240 and CBS 2954, CBS 7240 and CBS 2986, CBS 7240 and CBS 5156, CBS 7240 and CBS 6017, CBS 7240 and yHDPN 421, CBS 7240 and yHKS 210, CBS 7240 and yHKS 212, CBS 7240 and yHKS 509, CBS 7240 and yHRVM 107, CBS 7240 and NCYC 966, and CBS 7240 and CBS 1483; NCYC 1526 and NCYC 1262, NCYC 1526 and NCYC 227, NCYC 1526 and NCYC 699, NCYC 1526 and NCYC 487, NCYC 1526 and NCYC 2340, NCYC 1526 and NCYC 1296, NCYC 1526 and NCYC 228, NCYC 1526 and NCYC 534, NCYC 1526 and NCYC 2801, NCYC 1526 and NCYC 1365, NCYC 1526 and NCYC 668, NCYC 1526 and NCYC 450, NCYC 1526 and NCYC 1236, NCYC 1526 and NCYC 75, NCYC 1526 and NCYC 112, NCYC 1526 and NCYC 115, NCYC 1526 and NCYC 177, NCYC 1526 and NCYC 223, NCYC 1526 and NCYC 224, NCYC 1526 and NCYC 240, NCYC 1526 and NCYC 340, NCYC 1526 and NCYC 478, NCYC 1526 and NCYC 479, NCYC 1526 and NCYC 510, NCYC 1526 and NCYC 530, NCYC 1526 and NCYC 965, NCYC1526 and NCYC 969, NCYC 1526 and NCYC 975, NCYC 1526 and NCYC 987, NCYC 1526 and NCYC 989, NCYC 1526 and NCYC 1322, NCYC 1526 and NCYC 1323, NCYC 1526 and NCYC 229, NCYC 1526 and NCYC 230, NCYC 1526 and NCYC 242, NCYC 1526 and NCYC 392, NCYC 1526 and NCYC 397, NCYC 1526 and NCYC 398, NCYC 1526 and NCYC 511, NCYC 1526 and NCYC 584, NCYC 1526 and NCYC 3420, NCYC 1526 and CBS 12357, NCYC 1526 and CBS 7001, NCYC 1526 and CBS 1174, NCYC 1526 and CBS 1177, NCYC 1526 and CBS 1245, NCYC 1526 and CBS 1386, NCYC 1526 and CBS 1488, NCYC 1526 and CBS 1502, NCYC 1526 and CBS 1504, NCYC 1526 and CBS 1548, NCYC 1526 and CBS 1550, NCYC 1526 and CBS 1551, NCYC 1526 and CBS 1552, NCYC 1526 and CBS 1603, NCYC 1526 and CBS 1605, NCYC 1526 and CBS 1606, NCYC 1526 and CBS 1608, NCYC 1526 and CBS 1665, NCYC 1526 and CBS 2165, NCYC 1526 and CBS 2442, NCYC 1526 and CBS 2945, NCYC 1526 and CBS 2954, NCYC 1526 and CBS 2986, NCYC 1526 and CBS 5156, NCYC 1526 and CBS 6017, NCYC 1526 and yHDPN 421, NCYC 1526 and yHKS 210, NCYC 1526 and yHKS 212, NCYC 1526 and yHKS 509, NCYC 1526 and yHRVM 107, NCYC 1526 and NCYC 966, and NCYC 1526 and CBS 1483; NCYC 1544 and NCYC 1262, NCYC 1544 and NCYC 227, NCYC1544 and NCYC 699, NCYC 1544 and NCYC 487, NCYC 1544 and NCYC 2340, NCYC 1544 and NCYC 1296, NCYC 1544 and NCYC 228, NCYC 1544 and NCYC 534, NCYC 1544 and NCYC 2801, NCYC 1544 and NCYC 1365, NCYC 1544 and NCYC 668, NCYC 1544 and NCYC 450, NCYC 1544 and NCYC 1236, NCYC 1544 and NCYC 75, NCYC 1544 and NCYC 112, NCYC 1544 and NCYC 115, NCYC 1544 and NCYC 177, NCYC 1544 and NCYC 223, NCYC 1544 and NCYC 224, NCYC 1544 and NCYC 240, NCYC 1544 and NCYC 340, NCYC 1544 and NCYC 478, NCYC 1544 and NCYC 479, NCYC 1544 and NCYC 510, NCYC 1544 and NCYC 530, NCYC 1544 and NCYC 965, NCYC 1544 and NCYC 969, NCYC 1544 and NCYC 975, NCYC 1544 and NCYC 987, NCYC 1544 and NCYC 989, NCYC 1544 and NCYC 1322, NCYC 1544 and NCYC 1323, NCYC 1544 and NCYC 229, NCYC 1544 and NCYC 230, NCYC 1544 and NCYC 242, NCYC 1544 and NCYC 392, NCYC 1544 and NCYC 397, NCYC 1544 and NCYC 398, NCYC 1544 and NCYC 511, NCYC 1544 and NCYC 584, NCYC 1544 and NCYC 3420, NCYC 1544 and CBS 12357, NCYC 1544 and CBS 7001, NCYC 1544 and CBS 1174, NCYC 1544 and CBS 1177, NCYC 1544 and CBS 1245, NCYC 1544 and CBS 1386, NCYC 1544 and CBS 1488, NCYC 1544 and CBS 1502, NCYC 1544 and CBS 1504, NCYC 1544 and CBS 1548, NCYC 1544 and CBS1550, NCYC 1544 and CBS 1551, NCYC 1544 and CBS 1552, NCYC 1544 and CBS 1603, NCYC 1544 and CBS 1605, NCYC 1544 and CBS 1606, NCYC 1544 and CBS 1608, NCYC 1544 and CBS 16 65, NCYC 1544 and CBS 2165, NCYC 1544 and CBS 2442, NCYC 1544 and CBS 2945, NCYC 1544 and CBS 2954, NCYC 1544 and CBS 2986, NCYC 1544 and CBS 5156, NCYC 1544 and CBS 6017, NCYC 1544 and yHDPN 421, NCYC 1544 and yHKS 210, NCYC 1544 and yHKS 212, NCYC 1544 and yHKS 509, NCYC 1544 and yHRVM 107, NCYC 1544 and NCYC 966, and NCYC 1544 and CBS 1483; 2359 and NCYC 1262, NCYC 2359 and NCYC 227, NCYC 2359 and NCYC 699, NCYC 2359 and NCYC 487, NCYC 2359 and NCYC 2340, NCYC 2359 and NCYC 1296, NCYC 2359 and NCYC 228, NCYC 2359 and NCYC 534, NCYC 2359 and NCYC 2801, NCYC 2359 and NCYC 1365, NCYC 2359 and NCYC 668, NCYC 2359 and NCYC 450, NCYC 2359 and NCYC 1236, NCYC 2359 and NCYC 75, NCYC 2359 and NCYC 112, NCYC 2359 and NCYC 115, NCYC 2359 and NCYC 177, NCYC 2359 and NCYC 223, NCYC 2359 and NCYC 224, NCYC 2359 and NCYC 240, NCYC 2359 and NCYC 340, NCYC 2359 and NCYC 478, NCYC 2359 and NCYC 479, NCYC 2359 and NCYC 510, NCYC 2359 and NCYC 530, NCYC 2359 and NCYC 965, NCYC 2359 and NCYC 969, NCYC 2359 and NCYC 975, NCYC 2359 and NCYC 987, NCYC 2359 and NCYC 989, NCYC 2359 and NCYC 1322, NCYC 2359 and NCYC 1323, NCYC 2359 and NCYC 229, NCYC 2359 and NCYC 230, NCYC 2359 and NCYC 242, NCYC2359 and NCYC 392, NCYC 2359 and NCYC 397, NCYC 2359 and NCYC 398, NCYC 2359 and NCYC 511, NCYC 2359 and NCYC 584, NCYC 2359 and NCYC 3420, NCYC 2359 and CBS 12357, NCYC 2359 and CBS 7001, NCYC 2359 and CBS 1174, NCYC 2359 and CBS 1177, NCYC 2359 and CBS 1245, NCYC 2359 and CBS 1386, NCYC 2359 and CBS 1488, NCYC 2359 and CBS 1502, NCYC 2359 and CBS 1504, NCYC 2359 and CBS 1548, NCYC 2359 and CBS 1550, NCYC 2359 and CBS 1551, NCYC 2359 and CBS 1552, NCYC 2359 and CBS 1603, NCYC 2359 and CBS 1605, NCYC 2359 and CBS 1606, NCYC 2359 and CBS 1608, NCYC 2359 and CBS 1665, NCYC 2359 and CBS 2165, NCYC 2359 and CBS 2442, NCYC 2359 and CBS 2945, NCYC 2359 and CBS 2954, NCYC 2359 and CBS 2986, NCYC 2359 and CBS 5156, NCYC 2359 and CBS 6017, NCYC 2359 and yHDPN 421, NCYC 2359 and yHKS 210, NCYC 2359 and yHKS 212, NCYC 2359 and yHKS 509, NCYC 2359 and yHRVM 107, NCYC 2359 and NCYC 966, and NCYC 2359 and CBS 1483; NCYC 986 and NCYC 1262, NCYC 986 and NCYC 227, NCYC 986 and NCYC 699, NCYC 986 and NCYC 487, NCYC 986 and NCYC 2340, NCYC 986 and NCYC 1296, NCYC 986 and NCYC 228, NCYC 986 and NCYC 534, NCYC 986 and NCYC 2801, NCYC 986 and NCYC 1365, NCYC 986 and NCYC 668, NCYC 986 and NCYC 450, NCYC986 and NCYC 1236, NCYC 986 and NCYC 75, NCYC 986 and NCYC 112, NCYC 986 and NCYC 115, NCYC 986 and NCYC 177, NCYC 986 and NCYC 223, NCYC 986 and NCYC 224, NCYC 986 and NCYC 240, NCYC 986 and NCYC 340, NCYC 986 and NCYC 478, NCYC 986 and NCYC 479, NCYC 986 and NCYC 510, NCYC 986 and NCYC 530, NCYC 986 and NCYC 965, NCYC 986 and NCYC 969, NCYC 986 and NCYC 975, NCYC 986 and NCYC 987, NCYC 986 and NCYC 989, NCYC 986 and NCYC 1322, NCYC 986 and NCYC 1323, NCYC 986 and NCYC 229, NCYC 986 and NCYC 230, NCYC 986 and NCYC 242, NCYC 986 and NCYC 392, NCYC 986 and NCYC 397, NCYC 986 and NCYC 398, NCYC 986 and NCYC 511, NCYC 986 and NCYC 584, NCYC 986 and NCYC 3420, NCYC 986 and CBS 12357, NCYC 986 and CBS 7001, NCYC 986 and CBS 1174, NCYC 986 and CBS 1177, NCYC 986 and CBS 1245, NCYC 986 and CBS 1386, NCYC 986 and CBS 1488, NCYC 986 and CBS 1502, NCYC 986 and CBS 1504, NCYC 986 and CBS 1548, NCYC 986 and CBS 1550, NCYC 986 and CBS 1551, NCYC 986 and CBS 1552, NCYC 986 and CBS 1603, NCYC 986 and CBS 1605, NCYC 986 and CBS 1606, NCYC 986 and CBS 1608, NCYC 986 and CBS 1665, NCYC 986 and CBS 2165, NCYC 986 and CBS 2442, NCYC 986 and CBS 2945, NCYC 986 and CBS 2954, NCYC 986 and CBS 2986, NCYC 986 and CBS5156, NCYC 986 and CBS 6017, NCYC 986 and yHDPN 421, NCYC 986 and yHKS 210, NCYC 986 and yHKS 212, NCYC 986 and yHKS 509, NCYC 986 and yHRVM 107, NCYC 986 and NCYC 966, and NCYC 986 and CBS 1483; NCYC 1295 and NCYC 1262, NCYC 1295 and NCYC 227, NCYC 1295 and NCYC 699, NCYC 1295 and NCYC 487, NCYC 1295 and NCYC 2340, NCYC 1295 and NCYC 1296, NCYC 1295 and NCYC 228, NCYC 1295 and NCYC 534, NCYC 1295 and NCYC 2801, NCYC 1295 and NCYC 1365, NCYC 1295 and NCYC 668, NCYC 1295 and NCYC 450, NCYC 1295 and NCYC 1236, NCYC 1295 and NCYC 75, NCYC 1295 and NCYC 112, NCYC 1295 and NCYC 115, NCYC 1295 and NCYC 177, NCYC 1295 and NCYC 223, NCYC 1295 and NCYC 224, NCYC 1295 and NCYC 240, NCYC 1295 and NCYC 340, NCYC 1295 and NCYC 478, NCYC 1295 and NCYC 479, NCYC 1295 and NCYC 510, NCYC 1295 and NCYC 530, NCYC 1295 and NCYC 965, NCYC 1295 and NCYC 969, NCYC 1295 and NCYC 975, NCYC 1295 and NCYC 987, NCYC 1295 and NCYC 989, NCYC 1295 and NCYC 1322, NCYC 1295 and NCYC 1323, NCYC 1295 and NCYC 229, NCYC 1295 and NCYC 230, NCYC 1295 and NCYC 242, NCYC 1295 and NCYC 392, NCYC 1295 and NCYC 397, NCYC 1295 and NCYC 398, NCYC 1295 and NCYC 511, NCYC 1295 and NCYC 584, NCYC 1295 and NCYC 3420, NCYC1295 and CBS 12357, NCYC 1295 and CBS 7001, NCYC 1295 and CBS 1174, NCYC 1295 and CBS 1177, NCYC 1295 and CBS 1245, NCYC 1295 and CBS 1386, NCYC 1295 and CBS 1488, NCYC 1295 and CBS 1502, NCYC 1295 and CBS 1504, NCYC 1295 and CBS 1548, NCYC 1295 and CBS 1550, NCYC 1295 and CBS 1551, NCYC 1295 and CBS 1552, NCYC 1295 and CBS 1603, NCYC 1295 and CBS 1605, NCYC 1295 and CBS 1606, NCYC 1295 and CBS 1608, NCYC 1295 and CBS 1665, NCYC 1295 and CBS 2165, NCYC 1295 and CBS 2442, NCYC 1295 and CBS 2945, NCYC 1295 and CBS 2954, NCYC 1295 and CBS 2986, NCYC 1295 and CBS 5156, NCYC 1295 and CBS 6017, NCYC 1295 and yHDPN 421, NCYC 1295 and yHKS 210, NCYC 1295 and yHKS 212, NCYC 1295 and yHKS 509, NCYC 1295 and yHRVM 107, NCYC 1295 and NCYC 966, and NCYC 1295 and CBS 1483; NCYC 1516 and NCYC 1262, NCYC 1516 and NCYC 227, NCYC 1516 and NCYC 699, NCYC 1516 and NCYC 487, NCYC 1516 and NCYC 2340, NCYC 1516 and NCYC 1296, NCYC 1516 and NCYC 228, NCYC 1516 and NCYC 534, NCYC 1516 and NCYC 2801, NCYC 1516 and NCYC 1365, NCYC 1516 and NCYC 668, NCYC 1516 and NCYC 450, NCYC 1516 and NCYC 1236, NCYC 1516 and NCYC 75, NCYC 1516 and NCYC 112, NCYC 1516 and NCYC 115, NCYC 1516 and NCYC 177, NCYC1516 and NCYC 223, NCYC 1516 and NCYC 224, NCYC 1516 and NCYC 240, NCYC 1516 and NCYC 340, NCYC 1516 and NCYC 478, NCYC 1516 and NCYC 479, NCYC 1516 and NCYC 510, NCYC 1516 and NCYC 530, NCYC 1516 and NCYC 965, NCYC 1516 and NCYC 969, NCYC 1516 and NCYC 975, NCYC 1516 and NCYC 987, NCYC 1516 and NCYC 989, NCYC 1516 and NCYC 1322, NCYC 1516 and NCYC 1323, NCYC 1516 and NCYC 229, NCYC 1516 and NCYC 230, NCYC 1516 and NCYC 242, NCYC 1516 and NCYC 392, NCYC 1516 and NCYC 397, NCYC 1516 and NCYC 398, NCYC 1516 and NCYC 511, NCYC 1516 and NCYC 584, NCYC 1516 and NCYC 3420, NCYC 1516 and CBS 12357, NCYC 1516 and CBS 7001, NCYC 1516 and CBS 1174, NCYC 1516 and CBS 1177, NCYC 1516 and CBS 1245, NCYC 1516 and CBS 1386, NCYC 1516 and CBS 1488, NCYC 1516 and CBS 1502, NCYC 1516 and CBS 1504, NCYC 1516 and CBS 1548, NCYC 1516 and CBS 1550, NCYC 1516 and CBS 1551, NCYC 1516 and CBS 1552, NCYC 1516 and CBS 1603, NCYC 1516 and CBS 1605, NCYC 1516 and CBS 1606, NCYC 1516 and CBS 1608, NCYC 1516 and CBS 1665, NCYC 1516 and CBS 2165, NCYC 1516 and CBS 2442, NCYC 1516 and CBS 2945, NCYC 1516 and CBS 2954, NCYC 1516 and CBS 2986, NCYC 1516 and CBS 5156, NCYC 1516 and CBS 6017, NCYC 1516 and yHDPN 421, NCYC 1516 and yHKS 210, NCYC 1516 and yHKS 212, NCYC 1516 and yHKS 509, NCYC 1516 and yHRVM 107, NCYC 1516 and NCYC 966, and NCYC 1516 and CBS 1483;1326 and NCYC 1262, NCYC 1326 and NCYC 227, NCYC 1326 and NCYC 699, NCYC 1326 and NCYC 487, NCYC 1326 and NCYC 2340, NCYC 1326 and NCYC 1296, NCYC 1326 and NCYC 228, NCYC 1326 and NCYC 534, NCYC 1326 and NCYC 2801, NCYC 1326 and NCYC 1365, NCYC 1326 and NCYC 668, NCYC 1326 and NCYC 450, NCYC 1326 and NCYC 1236, NCYC 1326 and NCYC 75, NCYC 1326 and NCYC 112, NCYC 1326 and NCYC 115, NCYC 1326 and NCYC 177, NCYC 1326 and NCYC 223, NCYC 1326 and NCYC 224, NCYC 1326 and NCYC 240, NCYC 1326 and NCYC 340, NCYC 1326 and NCYC 478, NCYC 1326 and NCYC 479, NCYC 1326 and NCYC 510, NCYC 1326 and NCYC 530, NCYC 1326 and NCYC 965, NCYC 1326 and NCYC 969, NCYC 1326 and NCYC 975, NCYC 1326 and NCYC 987, NCYC 1326 and NCYC 989, NCYC 1326 and NCYC 1322, NCYC 1326 and NCYC 1323, NCYC 1326 and NCYC 229, NCYC 1326 and NCYC 230, NCYC 1326 and NCYC 242, NCYC 1326 and NCYC 392, NCYC 1326 and NCYC 397, NCYC 1326 and NCYC 398, NCYC 1326 and NCYC 511, NCYC 1326 and NCYC 584, NCYC 1326 and NCYC 3420, NCYC 1326 and CBS 12357, NCYC 1326 and CBS 7001, NCYC 1326 and CBS 1174, NCYC 1326 and CBS 1177, NCYC 1326 and CBS 1245, NCYC 1326 and CBS 1386, NCYC 1326 and CBS 1488, NCYC 1326 and CBS 1502, NCYC 1326 and CBS1504, NCYC 1326 and CBS 1548, NCYC 1326 and CBS 1550, NCYC 1326 and CBS 1551, NCYC 1326 and CBS 1552, NCYC 1326 and CBS 1603, NCYC 1326 and CBS 1605, NCYC 1326 and CBS 1606, NCYC 1326 and CBS 1608, NCYC 1326 and CBS 1665, NCYC 1326 and CBS 2165, NCYC 1326 and CBS 2442, NCYC 1326 and CBS 2945, NCYC 1326 and CBS 2954, NCYC 1326 and CBS 2986, NCYC 1326 and CBS 5156, NCYC 1326 and CBS 6017, NCYC 1326 and yHDPN 421, NCYC 1326 and yHKS 210, NCYC 1326 and yHKS 212, NCYC 1326 and yHKS 509, NCYC 1326 and yHRVM 107, NCYC 1326 and NCYC 966, and NCYC 1326 and CBS 1483; NCYC 2398 and NCYC 1262, NCYC 2398 and NCYC 227, NCYC 2398 and NCYC 699, NCYC 2398 and NCYC 487, NCYC 2398 and NCYC 2340, NCYC 2398 and NCYC 1296, NCYC 2398 and NCYC 228, NCYC 2398 and NCYC 534, NCYC 2398 and NCYC 2801, NCYC 2398 and NCYC 1365, NCYC 2398 and NCYC 668, NCYC 2398 and NCYC 450, NCYC 2398 and NCYC 1236, NCYC 2398 and NCYC 75, NCYC 2398 and NCYC 112, NCYC 2398 and NCYC 115, NCYC 2398 and NCYC 177, NCYC 2398 and NCYC 223, NCYC 2398 and NCYC 224, NCYC 2398 and NCYC 240, NCYC 2398 and NCYC 340, NCYC 2398 and NCYC 478, NCYC 2398 and NCYC 479, NCYC 2398 and NCYC 510, NCYC 2398 and NCYC 530, NCYC 2398 and NCYC 965, NCYC2398 and NCYC 969, NCYC 2398 and NCYC 975, NCYC 2398 and NCYC 987, NCYC 2398 and NCYC 989, NCYC 2398 and NCYC 1322, NCYC 2398 and NCYC 1323, NCYC 2398 and NCYC 229, NCYC 2398 and NCYC 230, NCYC 2398 and NCYC 242, NCYC 2398 and NCYC 392, NCYC 2398 and NCYC 397, NCYC 2398 and NCYC 398, NCYC 2398 and NCYC 511, NCYC 2398 and NCYC 584, NCYC 2398 and NCYC 3420, NCYC 2398 and CBS 12357, NCYC 2398 and CBS 7001, NCYC 2398 and CBS 1174, NCYC 2398 and CBS 1177, NCYC 2398 and CBS 1245, NCYC 2398 and CBS 1386, NCYC 2398 and CBS 1488, NCYC 2398 and CBS 1502, NCYC 2398 and CBS 1504, NCYC 2398 and CBS 1548, NCYC 2398 and CBS 1550, NCYC 2398 and CBS 1551, NCYC 2398 and CBS 1552, NCYC 2398 and CBS 1603, NCYC 2398 and CBS 1605, NCYC 2398 and CBS 1606, NCYC 2398 and CBS 1608, NCYC 2398 and CBS 1665, NCYC 2398 and CBS 2165, NCYC 2398 and CBS 2442, NCYC 2398 and CBS 2945, NCYC 2398 and CBS 2954, NCYC 2398 and CBS 2986, NCYC 2398 and CBS 5156, NCYC 2398 and CBS 6017, NCYC 2398 and yHDPN 421, NCYC 2398 and yHKS 210, NCYC 2398 and yHKS 212, NCYC 2398 and yHKS 509, NCYC 2398 and yHRVM 107, NCYC 2398 and NCYC 966, and NCYC 2398 and CBS 1483; NCYC 669 and NCYC 1262, NCYC 669 and NCYC 227, NCYC 669 and NCYC699, NCYC 669 and NCYC 487, NCYC 669 and NCYC 2340, NCYC 669 and NCYC 1296, NCYC 669 and NCYC 228, NCYC 669 and NCYC 534, NCYC 669 and NCYC 2801, NCYC 669 and NCYC 1365, NCYC 669 and NCYC 668, NCYC 669 and NCYC 450, NCYC 669 and NCYC 1236, NCYC 669 and NCYC 75, NCYC 669 and NCYC 112, NCYC 669 and NCYC 115, NCYC 669 and NCYC 177, NCYC 669 and NCYC 223, NCYC 669 and NCYC 224, NCYC 669 and NCYC 240, NCYC 669 and NCYC 340, NCYC 669 and NCYC 478, NCYC 669 and NCYC 479, NCYC 669 and NCYC 510, NCYC 669 and NCYC 530, NCYC 669 and NCYC 965, NCYC 669 and NCYC 969, NCYC 669 and NCYC 975, NCYC 669 and NCYC 987, NCYC 669 and NCYC 989, NCYC 669 and NCYC 1322, NCYC 669 and NCYC 1323, NCYC 669 and NCYC 229, NCYC 669 and NCYC 230, NCYC 669 and NCYC 242, NCYC 669 and NCYC 392, NCYC 669 and NCYC 397, NCYC 669 and NCYC 398, NCYC 669 and NCYC 511, NCYC 669 and NCYC 584, NCYC 669 and NCYC 3420, NCYC 669 and CBS 12357, NCYC 669 and CBS 7001, NCYC 669 and CBS 1174, NCYC 669 and CBS 1177, NCYC 669 and CBS 1245, NCYC 669 and CBS 1386, NCYC 669 and CBS 1488, NCYC 669 and CBS 1502, NCYC 669 and CBS 1504, NCYC 669 and CBS 1548, NCYC 669 and CBS 1550, NCYC 669 and CBS 1551, NCYC 669 and CBS 1552, NCYC 669 and CBS1603, NCYC 669 and CBS 1605, NCYC 669 and CBS 1606, NCYC 669 and CBS 1608, NCYC 669 and CBS 1665, NCYC 669 and CBS 2165, NCYC 669 and CBS 2442, NCYC 669 and and CBS 2945, NCYC 669 and CBS 2954, NCYC 669 and CBS 2986, NCYC 669 and CBS 5156, NCYC 669 and CBS 6017, NCYC 669 and yHDPN 421, NCYC 669 and yHKS 210, NCYC 669 and yHKS 212, NCYC 669 and yHKS 509, NCYC 669 and yHRVM 107, NCYC 669 and NCYC 966, and NCYC 669 and CBS 1483; NCYC 2921 and NCYC 1262, NCYC 2921 and NCYC 227, NCYC 2921 and NCYC 699, NCYC 2921 and NCYC 487, NCYC 2921 and NCYC 2340, NCYC 2921 and NCYC 1296, NCYC 2921 and NCYC 228, NCYC 2921 and NCYC 534, NCYC 2921 and NCYC 2801, NCYC 2921 and NCYC 1365, NCYC 2921 and NCYC 668, NCYC 2921 and NCYC 450, NCYC 2921 and NCYC 1236, NCYC 2921 and NCYC 75, NCYC 2921 and NCYC 112, NCYC 2921 and NCYC 115, NCYC 2921 and NCYC 177, NCYC 2921 and NCYC 223, NCYC 2921 and NCYC 224, NCYC 2921 and NCYC 240, NCYC 2921 and NCYC 340, NCYC 2921 and NCYC 478, NCYC 2921 and NCYC 479, NCYC 2921 and NCYC 510, NCYC 2921 and NCYC 530, NCYC 2921 and NCYC 965, NCYC 2921 and NCYC 969, NCYC 2921 and NCYC 975, NCYC 2921 and NCYC 987, NCYC 2921 and NCYC 989, NCYC 2921 and NCYC 1322, NCYC 2921 and NCYC 1323, NCYC 2921 and NCYC 229, NCYC 2921 and NCYC 230, NCYC 2921 and NCYC 242, NCYC 2921 and NCYC 392, NCYC 2921 and NCYC 397, NCYC 2921 and NCYC 398, NCYC2921 and NCYC 511, NCYC 2921 and NCYC 584, NCYC 2921 and NCYC 3420, NCYC 2921 and CBS 12357, NCYC 2921 and CBS 7001, NCYC 2921 and CBS 1174, NCYC 2921 and CBS 1177, NCYC 2921 and CBS 1245, NCYC 2921 and CBS 1386, NCYC 2921 and CBS 1488, NCYC 2921 and CBS 1502, NCYC 2921 and CBS 1504, NCYC 2921 and CBS 1548, NCYC 2921 and CBS 1550, NCYC 2921 and CBS 1551, NCYC 2921 and CBS 1552, NCYC 2921 and CBS 1603, NCYC 2921 and CBS 1605, NCYC 2921 and CBS 1606, NCYC 2921 and CBS 1608, NCYC 2921 and CBS 1665, NCYC 2921 and CBS 2165, NCYC 2921 and CBS 2442, NCYC 2921 and CBS 2945, NCYC 2921 and CBS 2954, NCYC 2921 and CBS 2986, NCYC 2921 and CBS 5156, NCYC 2921 and CBS 6017, NCYC 2921 and yHDPN 421, NCYC 2921 and yHKS 210, NCYC 2921 and yHKS 212, NCYC 2921 and yHKS 509, NCYC 2921 and yHRVM 107, NCYC 2921 and NCYC 966, and NCYC 2921 and CBS 1483.

[0088] In one embodiment, such yeast combinations do not include brewer's yeast, baker's yeast, or brewer's yeast and baker's yeast.

[0089] Such yeast may further comprise one or more naturally occurring mutations, and / or mutations resulting from mutagenesis, in at least one of the PAD1 and FDC1 genes, a gene involved in the transcriptional regulation of at least one of such genes, and / or a gene encoding a protein involved in the uptake of phenolic acids, preferably ferulic acid, or a protein involved in the transport of decarboxylated phenolic compounds, preferably 4-vinylguaiacol, and / or a gene involved in the transcriptional regulation of such genes.

[0090] A combination of such yeast strains differing in their specificity for fermenting maltose, maltotriose, fructose and / or glucose present in the wort to ethanol may be added to the wort at the same time at the start of fermentation or may be added sequentially to the wort at different times.

[0091] When large amounts of wort are to be fermented, the yeast strains may be pre-cultured to obtain sufficient amounts of each of the yeast strains to be added to the wort, either together or sequentially. The pre-culture of the individual strains is preferably carried out in a specific defined medium or in a non-defined medium such as wort. This pre-culture can be carried out with the separated strains individually or in combination. When pre-culturing a combination of yeast strains with different specificities for the fermentation of maltose, maltotriose, fructose and / or glucose, care must be taken that the ratio in which the at least two yeast strains with different specificities for the fermentation of maltose, maltotriose, fructose and / or glucose are present does not change during the pre-culture. This can be done, for example, by using a culture medium with different proportions of sugars selected from maltose, maltotriose, fructose and / or glucose. If one of the at least two yeast strains with different specificities for the fermentation of maltose, maltotriose, fructose and / or glucose may predominate, the combination may be cultured in a medium containing more of the sugar preferred by the non-predominant strain or strains.

[0092] Yeast strains that are specialists on one of maltose, maltotriose, fructose or glucose are preferably cultured in a synthetic medium containing the particular sugar that the yeast strain prefers.

[0093] Such pre-culture may be carried out in several steps. For example, dried or frozen stocks from at least two yeast strains with different specificities for the fermentation of maltose, maltotriose, fructose and / or glucose may each be inoculated onto a medium or plate containing such specific sugars to generate a "working" master stock. Such a master stock may be grown in increasing culture sizes until a sufficient amount of yeast is produced to be passed onto a propagation plant. The number of passes depends on the amount of yeast strain required to inoculate the wort in the propagation plant, but is generally kept to a minimum to reduce the risk of infection.

[0094] Such pre-cultures may include a counter-selection step in which each batch of specialist yeast strain is inoculated onto a medium containing a combination of the two remaining sugars, and if the specialist yeast strain shows enhanced growth on the counter-selection medium containing the other two sugars, such batches may be discarded.

[0095] The inoculation of the wort in the propagation plant with at least two yeast strains having different specificities for the fermentation of maltose, maltotriose, fructose and / or glucose can be carried out simultaneously or separated in time, in a ratio of 20:1 to 1:20 for each of the at least two yeast strains having different specificities for the fermentation of maltose, maltotriose, fructose and / or glucose.

[0096] In a preferred embodiment, the wort in the propagation plant is inoculated with two yeast strains having different specificities for the fermentation of maltose, maltotriose, fructose and / or glucose in a numerical ratio of 20:1, 10:1, 8:1, 5:1, 4:1, 3:1, 2:1 or 1:1.

[0097] Such inoculation of the wort in a propagation plant with two yeast strains having different specificities for the fermentation of maltose, maltotriose, fructose and / or glucose in a ratio based on numbers of 20:1, 10:1, 8:1, 5:1, 4:1, 3:1, 2:1 or 1:1 can be carried out at the beginning of the fermentation or at different times during the fermentation. For example, the fermentation can be started with a yeast strain with a preference for maltotriose at time zero, and then the yeast strain with a preference for maltose can be added after a certain time, for example after 1 day, 2 days, 3 days, 5 days. Starting the fermentation with only one of the yeast strains with a preference for the fermentation of maltose, maltotriose, fructose or glucose can have the advantage that this strain is not overgrown by the other strains. However, as will be appreciated by those skilled in the art, inoculating yeast strains with different specificities for fermenting maltose, maltotriose, fructose and / or glucose at different times is equivalent to inoculating yeast strains with different specificities for fermenting maltose, maltotriose, fructose and / or glucose together at the start of fermentation, provided that one yeast strain used for inoculation on day 0 is in excess over one or more other yeast strains. EXAMPLES

[0098] Example 1 Materials and Methods Stocks of 143 yeast collection institutions Freeze-dried cells from glass test tubes of strains obtained from public yeast collections (see Table 1) were suspended in sterile water, and 100 μl of the cell resuspension was then plated onto YPD agar plates (10 g L -1 , Bacto Yeast Extract, 20 g L -1 Bacto peptone, 20 g L -1 Glucose and 20 g L -1 The plates were incubated at 20°C for 5 days, after which one colony per strain was picked and plated on 35 ml of YPD medium (10 gL -1 , Bacto Yeast Extract, 20gL -1 Bacto Peptone, 20gL -1For the remaining strains, agar slants containing individual colonies or pure glycerol stock cultures were directly inoculated into 500 ml round-bottom flasks filled with 35 ml YPD. Cultures were grown for 48 h in an Innovova 44 incubator shaker (Eppendorf, Nijmegen, The Netherlands) set at 20°C and 200 rpm. Finally, cultures were supplemented with 30% v / v glycerol and stored at -80°C.

[0099] Preparation of wort medium Wort at Plateau 16.5, corresponding to a fermentable sugar content of approximately 120 g / L, was spiked with 1.5 mg / L ZnSO4. The spiked wort was then autoclaved at 110 °C for 20 min. During this process, precipitated flocs were formed, likely due to protein aggregation. The sterile wort was stored at 4 °C, filtered using a 0.2 μm Nalgene Rapid-Flow bottle-top filter (Thermo Fisher Scientific, Waltham, Massachusetts, United States), and diluted 3-fold with sterile demineralized water to obtain a wort medium with a final fermentable sugar concentration of approximately 40 g / L. Prior to the experiment, the wort medium was supplemented with 1.2 ml / L of 20% Pluronic PE9200 antifoam (BASF, Ludwigshafen, Germany).

[0100] Microaerobic culture Microaerobic cultivation was performed as described previously (Brouwers et al., 2019. bioRxiv 679563; Brouwers et al., 2019. PLoS Genet 15:e1007853). All strains were tested in triplicate. The control strain CBS1483 was included in all experiments. One glycerol stock tube (approximately 1 ml) of each strain was inoculated into a 50 ml CELLSTAR cell culture tube (Greiner Bio-One, Kremsmunster, Austria) filled with 20 ml of wort medium. These precultures were incubated at a 45° angle in a shaker incubator set at 12 °C and 200 rpm for 3 days or until OD660 The precultures were incubated until the OD reached >8. The optical density at 660 nm was monitored using a 7200 Jenway Spectrometer (Jenway, Stone, UK). Each preculture was placed in three 250 mL sterile glass bottles containing 200 mL of wort medium and capped with a rubber septum. 660 A sterile cotton-filled needle was inserted into the septum of each flask to allow for the release of CO2 pressure that may build up during the culture. Overall, these conditions ensure that a microaerobic environment is maintained throughout the experiment. The vials were incubated at 12 °C and 200 rpm for approximately 5 days. Two samples were taken per day by piercing the septum with a needle and aspirating 2–5 ml with a sterile syringe. The OD for each sample was measured. 660 The pH of the samples was measured and each filtered sample was analyzed by high pressure liquid chromatography (HPLC) using an Agilent 1260 HPLC (Agilent Technologies, Santa Clara, CA) equipped with a Bio-Rad HPX 87 H column (Bio-Rad, Hercules, CA). -1 of H2SO4, 0.6 ml -1 The mixture was eluted at 60°C with a flow rate of 100 s. The metabolites were detected using an Agilent refractive index detector and an Agilent 1260 VWD detector.

[0101] result Analysis of the consumption rate ratio of maltose and maltotriose The consumption rate of maltose and maltotriose is expressed by the formula Y = Y0 * e (k*X)The M2 / M3 kinetic ratio was calculated by fitting an exponential curve of (k is the rate constant) to the time points of the exponential growth phase of the consumption curve (see Figure 1). The value of the slope of maltose was divided by the value of the slope of maltotriose to obtain the dimensionless value of the maltose / maltotriose (M2 / M3) kinetic ratio. The kinetic ratio was maximized to a value of 10. The distribution of the M2 / M3 kinetic ratio in all tested strains is shown in Figure 2. The values ​​of the M2 / M3 kinetic ratio ranged from 0.28 to 10 for all samples. The M2 / M3 kinetic ratio of the control strain CBS1483 ranged from 4 to 10. The M2 / M3 kinetic ratio of the strain CBS1513 (also known as NCYC396) ranged from 0.28 to 0.40. Finally, the M2 / M3 ratio of the strain WS34 / 70 ranged from 1.82 to 1.96.

[0102] Example 2 Fermentation of a consortium composed of two specialists (CBS 1483 and CBS 1513). Materials and Methods KK The yeast strains used in this study are shown in Table 2. Stock cultures were prepared in YPD (10 g L -1 Bacto Yeast Extract, 20 g L -1 Bacto peptone, 20 g L -1 The cultures were grown in 1000 ml of glucose at 20°C to early stationary phase, supplemented with sterile glycerol (final concentration 30% (v / v)) and stored in 1 mL aliquots at -80°C until further use. [Table 1]

[0103] Preparation of culture medium Very high gravity (VHG) wort was prepared with its desired apparent extract (AE) by the addition of 6.6 mg ZnSO4·7H2O per liter and 11.63 g D-maltose monohydrate (Glentham Life Sciences Ltd, Wiltshire, UK) and 3.80 g commercial mixed sugar (Dried Glucose syrup C plus 01987, Cargill, Haubourdin, France) per liter additional degree plateau (°P) to industrial wort for lager beer production (obtained from HEINEKEN Supply Chain BV, Zoeterwoude, The Netherlands) prepared by the infusion mash process including settling with β-amylase and α-amylase from barley malt. The VHG wort was autoclaved at 110°C for 20 min.

[0104] Tall tube characterization Characterization of yeast strains and different culture combinations was performed using 3 L stainless steel tall tubes (EBC Analytica Microbiologica (1977) Journal of the Institute of Brewing 83: 109-118) equipped with a water-cooling jacket (Leemberg, Zwijndrecht, The Netherlands) for a working volume of 2.25 L. Strains were characterized using both 20°P and 23°P VHG wort. Temperature was controlled at 12°C by a Lauda ECO RE 415 G cryostat (Lauda Dr. R. Wobser GmbH & Co., Lauda-Konigshofen, Germany). Precultures were inoculated from frozen stock cultures into 100 mL of YPD and incubated in an orbital incubator at 12°C and 200 rpm for approximately 3 days. Cultures from these shake flasks were used to inoculate 100 mL of YPM 6% (10 gL -1 Bacto Yeast Extract, 20gL -1 Bacto peptone, and 60gL -1Flasks containing 500 mL of 500 mM KOH (maltose) were inoculated to an optical density of 0.8 and grown at 12°C and 200 rpm for approximately 2 days. Cells from these precultures were harvested by centrifugation (4000 rpm, 10 min, 4°C), resuspended in demineralized water, and diluted at 5.10 6 cells mL -1 The VHG wort was inoculated with 100% yeast culture medium at a cell concentration of 1000 μg / ml. For mixed culture characterization, the different strains were grown in separate precultures and mixed in the desired ratios before inoculation. The inoculated VHG wort was stirred for 30 min before it was passaged into duplicate tall tubes. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8]

[0105] Analysis method CO2 evolution was measured continuously by a mass flow meter (Bronkhorst, Veenendaal, The Netherlands). Optical density was measured using a Libra S60 spectrophotometer (Biochrom Ltd., Cambridge, USA). Total cell concentration and viability were measured with a Nucleocounter YC-100 (Chemometec A / S, Allerod, Denmark). Apparent extract (AE) was measured using a handheld densitometer DMA 35 Basic (Anton Paar GmbH, Graz, Austria). HPLC analysis of sugar and metabolite concentrations was performed using 0.6 mL min of elution with 5 mM H2SO4. -1 Analysis was performed using an Agilent Infinity 1260 chromatography system (Agilent Technologies, Santa Clara, CA, USA) equipped with an Aminex HPX-87H column (BioRad Laboratories Inc., Lunteren, The Netherlands) at 60 ° C. The concentrations of esters, higher alcohols and vicinal diketones were measured on an Agilent 7890A series GC equipped with a DB-WAXetr column (Agilent) and a flame ionization detector (FID) for the analysis of esters and higher alcohols, and a CP-Sil8CB column (Agilent) and an electron capture detector (ECD) for the analysis of vicinal diketones.

[0106] result In tall tube experiments, the maltotriose specialist Saccharomyces pastorianus CBS1513 (also called S. carlsbergensis type strain ECHansen) and the maltose specialist S. pastorianus CBS1483 were tested separately in wort at 20° P to confirm the behavior of the sugar specialists. See Figure 3.

[0107] In proof-of-principle mixtures, CBS1483 and CBS1513 were tested in tall tubes with 20°P wort at ratios of 1:1, 4:1, and 1:4, as well as 10:1 and 1:10, and compared to the standard generalist WS34 / 70. Results show that the 1:1 and 4:1 (CBS1483:CB1513) mixtures reached fermentation to 2.5°P density 10 days prior to the 20°P W34 / 70 fermentation (Figure 3A). Residual maltotriose concentrations in the 1:1, 1:4, and 1:10 (CBS1483:CBS1513) mixtures were below 10 g / L after approximately 10 days, which was comparable to the maltotriose specialists. Residual maltotriose concentrations for W34 / 70 were approximately 20 g / L at 10 days, and for the maltose specialists, approximately 30 g / L at 10 days (Figure 3B). Residual maltose concentrations after 10 days of fermentation of the 1:1, 4:1, and 10:1 mixtures were approximately 5 g / L, although both WS34 / 70 and the maltotriose specialists reached these values ​​after 20 days, 10 days later (Figure 3C).

[0108] Ethanol production was accelerated in the 1:1, 4:1, and 10:1 (CBS1483:CBS1513) mixtures when compared to WS34 / 70 (Figure 3D), but the final amount of ethanol produced was comparable in WS34 / 70 and all mixtures (Figure 3E).

[0109] Further variables analyzed, including pH, CO2 production, glucose consumption, fructose consumption, and production of esters, higher alcohols, and vicinal diketones, did not vary significantly between the different cultures.

[0110] When tested in tall tubes with 2°P wort, comparable data were obtained for individual strains and mixtures. Results show that the 1:1 and 4:1 mixtures reached 2.5°P levels of attenuation sooner than the parent strains (Figure 4A). Residual maltotriose concentrations in the 1:1, 1:4 and 1:10 mixtures were comparable to the maltotriose specialist and lower than the WS34 / 70 and maltose specialist, whereas the residual maltose concentrations in the 1:1, 4:1 and 10:1 mixtures were comparable to the maltose specialist (Figure 4B).

[0111] Ethanol production was accelerated for the 4:1 mixture compared to the individual strains (data not shown), which was found to finish in approximately 3 / 4 the time required for the other strains.

[0112] Comparing the mixtures of strains with different substrate specificities for 20°P and 23°P, it can be seen that the 1:1 and 4:1 mixtures reach 2.5°P fermentability before the 23°P starting wort. In addition, the final ethanol production is about 20% higher when starting from the 23°P wort (see Figure 5A,B).

[0113] It is concluded that a mixture of strains with different substrate specificities, such as CBS1513 and CBS1483, results in faster ethanol production when compared to individual strains or the industry standard (W34 / 70).

[0114] Example 3 Materials and Methods Saccharomyces strains: Superstart™ and Thermosacc®, both kindly provided by Lallemand Biofuels & Distilled Spirits (Milwaukee, WI). Co-cultivation was carried out in 3 L stainless steel tall tubes with a working volume of 2.25 L in VHG wort at 23°P. Temperature was controlled at 12°C by a Lauda ECO RE415G cryostat. Total cell concentration and viability were measured with a Nucleocounter YC-100 (Chemometec A / S, Allerod, Denmark). Apparent extract (AE) was measured using a handheld density meter DMA 35 Basic (Anton Paar GmbH, Graz, Austria). HPLC analysis of sugar and ethanol concentrations was performed using 0.6 mL aliquots of 5 mM H2SO4 as eluent. -1 The chromatography was performed using an Agilent Infinity 1260 chromatography system (Agilent Technologies, Santa Clara, Calif., USA) equipped with an Aminex HPX-87H column (BioRad Laboratories Inc., Lunteren, The Netherlands) at 60° C. using a flow rate of 1000 s.p.m.

[0115] result To check whether a mixture of strains Superstart™ and Thermosacc® could efficiently consume a mixture of glucose, maltose and maltotriose from the wort as proposed in WO 2013 / 181496, a mixture of the two strains was used in a 1:1 ratio to inoculate a VHG wort at 23° P. These Saccharomyces cerevisiae strains are active dry yeasts for use in fuel ethanol and beverage alcohol fermentation. Superstart is a brewer's yeast selected for its rapid fermentation initiation and stress tolerance to temperature, pH and osmotic pressure. This strain has also been characterized as a maltotriose positive strain.

[0116] Thermosacc® is a brewer's Saccharomyces cerevisiae yeast selected for use in high-temperature, high-gravity fermentations, high sugar and high alcohol concentrations. It performs well at temperatures up to 38° C. and at alcohol concentrations above 20% by volume (16% by weight).

[0117] Fermentations were carried out at 12°C in 3L stainless steel tall tubes (EBC Analytica Microbiologica, 1977. J Institute Brewing 83:109-118) with a working volume of 2.25L and equipped with a water-cooled jacket (Leemberg, Zwijndrecht, The Netherlands). The results of the quadruplicate fermentations were compared with those of a mixture of specialists CBS1483:CB1513 in an initial ratio of 1:1 (Figure 4). As expected, the two commercial strains initially selected for efficient fermentation at high temperatures above 30°C did not ferment efficiently at low temperatures below 30°C. The results show that the 1:1 ratio of Supersart™ and Thermossac reached a fermentation degree of 15.5°P after 19 days (Figure 6A). At that time, the strain mixture was no longer metabolically active as indicated by the CO2 profile reaching the baseline (null) value (Figure 7). The concentrations of the individual sugars were 32% (44.8 g L−1) of maltose and maltotriose, respectively. -1 ) and 4.5% (4.5gL -1 ) was consumed (Figures 6B and 6C). This resulted in a decrease in ethanol concentration compared to the CBS1483-CBS1513 coculture (Figure 6D).

[0118] As a comparison under similar conditions, a 1:1 ratio CBS1483:CBS1513 mixture was able to reach values ​​below 7°C in 20 days and a fermentation degree of 2.5°P after 40 days (Figure 4). At day 19, the co-culture of CBS strains consumed 83% of the maltotriose and 76% of the maltose initially present (Figure 4).

[0119] These results showed that Superstart and Thermosacc were selected for their excellent fermentation properties but are not suitable for wort fermentation at low temperatures for producing fermented beverages such as beer, preferably lager beer.

Claims

1. A method for producing a fermented beverage, comprising: - providing wort, - adding hops and at least two fermenting yeast strains to the wort, whereby the at least two fermenting yeast strains differ in their substrate specificity for sugars in the wort, in particular for glucose, fructose, maltose and / or maltotriose; - incubating said wort with said at least two fermenting yeast strains for a period of time; - optionally removing said at least two fermenting yeast strains from said fermented wort, This method produces a fermented beverage.

2. 2. The method of claim 1, wherein the wort is a high gravity wort having a gravity greater than 16 degrees plateau.

3. 10. The method of claim 1, wherein the at least two fermenting yeast strains are added to the wort simultaneously or sequentially.

4. 2. The method of claim 1, wherein the fermented beverage has an apparent degree of fermentation of at least 0.

8.

5. 10. The method of claim 1, wherein fermentation of high gravity wort, such as above 16 degrees plateau, results in a fermented beverage having a final gravity of less than 2.5 degrees plateau within 15 days of fermentation.

6. 10. The method of claim 1, wherein the at least two fermenting yeast strains are a mixture of Saccharomyces cerevisiae, S. pastorianus, S. eubayanus yeast strains, and / or hybrids thereof.

7. 2. The method of claim 1, wherein the fermentation is carried out at a temperature of 6 to 25°C, preferably 8 to 15°C.

8. 2. The method of claim 1, wherein the fermented beverage is beer, preferably lager beer.

9. 2. The method of claim 1, wherein the fermenting yeast strain that prefers a maltose substrate is Saccharomyces pastorianus strain CBS1483.

10. 2. The method of claim 1, wherein the fermenting yeast strain that prefers a maltotriose substrate is S. pastorianus strain CBS1513.

11. A fermented beverage, preferably beer, more preferably lager beer, having a high alcohol content, such as more than 5% alcohol by volume (ABV) or more than 6% ABV.

12. A fermented beverage, preferably beer, more preferably lager beer, produced by the method according to any one of claims 1 to 10.

13. 1. Use of at least two fermenting yeast strains differing in substrate specificity for sugars in wort, in particular for glucose, fructose, maltose, and / or maltotriose, for the production of a fermented beverage, preferably beer, most preferably lager beer.

14. 14. The use according to claim 13, wherein the at least two fermenting yeast strains are added to the wort simultaneously or sequentially.

15. 15. The use according to claim 13 or 14, wherein the at least two fermenting yeast strains comprise a fermenting yeast strain that prefers a maltose substrate, such as Saccharomyces pastorianus strain CBS1483, a fermenting yeast strain that prefers a maltotriose substrate, such as S. pastorianus strain CBS1513, or a combination thereof.

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