US20140234480A1 - Enhancement of beer flavor by a combination of pichia yeast and different hop varieties - Google Patents

Enhancement of beer flavor by a combination of pichia yeast and different hop varieties Download PDF

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US20140234480A1
US20140234480A1 US14/241,761 US201214241761A US2014234480A1 US 20140234480 A1 US20140234480 A1 US 20140234480A1 US 201214241761 A US201214241761 A US 201214241761A US 2014234480 A1 US2014234480 A1 US 2014234480A1
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fermentation
beer
yeast strain
ethyl
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Sofie Sarens
Jan Hendrik Swiegers
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Chr Hansen AS
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
    • C12C11/00—Fermentation processes for beer
    • C12C11/003—Fermentation of beerwort
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
    • C12C12/00—Processes specially adapted for making special kinds of beer
    • C12C12/002—Processes specially adapted for making special kinds of beer using special microorganisms
    • C12C12/006—Yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
    • C12C12/00—Processes specially adapted for making special kinds of beer
    • C12C12/04—Beer with low alcohol content
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
    • C12C7/00—Preparation of wort
    • C12C7/20—Boiling the beerwort
    • C12C7/205—Boiling with hops
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
    • C12C7/00—Preparation of wort
    • C12C7/28—After-treatment, e.g. sterilisation
    • C12C7/287—Treating beerwort with hopextract

Definitions

  • the present invention relates to the field of beer brewing and enhancement of beer flavor. Specifically, the invention relates to a method of brewing beer comprising a step of fermentation of a hopped wort with a Pichia spp. (species), where there is an interaction of the hops with the Pichia spp. yeast strain to enhance the flavor of beer.
  • a Pichia spp. species
  • hops Another important factor is the hop.
  • bitter hops are used for lager beers to give extra bitterness to the beer.
  • Aroma hops are used for specialty beers to enhance the flavor.
  • starter cultures is a common practice in yeast industries.
  • a pure yeast strain often does not combine all optimal traits desired in brewing, as there are two major objectives for using specific yeast strains in the beer industry: improving the efficiency of the production process and obtaining a good quality of the final product (Saerens et al. 2010).
  • the most desirable flavor compounds in beer are esters and higher alcohols, giving a fruity aroma to the beer (Verstrepen et al. 2003).
  • the most important flavor-active esters in beer are acetate esters, such as ethyl acetate (“solvent”-like aroma) and isoamyl acetate (“banana” aroma), and ethyl esters, such as ethyl hexanoate and octanoate (“apple” aroma).
  • the most important higher alcohol is isoamyl alcohol (“banana” aroma).
  • brewers have distinguished two types of brewer's yeast: ale and lager yeast, according to their use for the production of ales and lagers, respectively.
  • Ale and lager brewer's yeasts belong to the genus Saccharomyces : S. cerevisiae (ale yeast) and S. pastorianus (lager yeast).
  • Lager yeast is used for the production of pilsner beers, which comprises 90% of the beers produced in the world.
  • WO 2009/110807 is related to yeast strains for use in fermentation processes and to a method of enhancing flavor in a product of fermentation by use of a non- Saccharomyces yeast strain.
  • WO 2009/110807 relates to fermentation of wine and does not mention use of non- Saccharomyces species in brewing of beer.
  • WO 2008/077986 describes a process for producing alcoholic beverages with increased and/or different aromatic terpene content by using genetically modified microorganisms expressing genes which code for monoterpene synthase during fermentation.
  • Pichia is mentioned in a list of theoretically possible yeast strains (together with other yeast strains such as e.g. Saccharomyces ) for fermenting alcoholic beverages (beer is mentioned as an example together with other alcoholic beverages such as wine, cava, champagne, cider and sake).
  • yeast strains such as e.g. Saccharomyces
  • German patent DD 288619 A5 describes the use of Pichia yeast to ferment beer wort. The beer wort is not added hops.
  • the problem to be solved by the present invention relates to the provision of a new method for brewing beer wherein the presence of desirable flavor compounds, such as esters and higher alcohols, is enhanced.
  • the solution is based on the surprising findings by the inventors that by using a method comprising fermenting hopped wort with a Pichia spp. yeast strain prior to or at the same time as fermentation of the hopped wort with a conventional brewer's yeast one is able to produce a beer with an improved flavor profile.
  • a first aspect of the invention relates to a method of brewing beer comprising the steps of:
  • a second aspect of the present invention relates to a beer obtainable by the method according to the first aspect of the invention.
  • a third aspect of the present invention relates to use of a Pichia species for enhancement of beer flavor.
  • a fourth aspect of the present invention relates to use of a Pichia species for brewing of beer.
  • FIG. 27 illustrates acetaldehyde concentrations in finished beers.
  • FIG. 28 illustrates ethyl acetate concentrations in finished beers.
  • FIG. 29 illustrates isoamylacetate concentrations in finished beers.
  • FIG. 30 illustrates ethyl propionate concentrations in finished beers.
  • FIG. 31 illustrates ethyl butyrate concentrations in finished beers.
  • FIG. 32 illustrates ethyl valerate concentrations in finished beers.
  • FIG. 33 illustrates ethyl hexanoate concentrations in finished beers.
  • FIG. 34 illustrates ethyl octanoate concentrations in finished beers.
  • FIG. 35 illustrates ethyl decanoated concentrations in finished beers.
  • FIG. 36 illustrates isobutanol concentrations in finished beers.
  • FIG. 37 illustrates isoamyl alcohol concentrations in finished beers.
  • FIG. 38 illustrates concentrations of the thiols 3-mercaptohexanol and 3-mercaptohexyl acetate in beer prepared with and without Pichia.
  • wort herein has the conventional meaning in the art and refers to the sugary liquid extracted from the mashing process of beer brewing.
  • beer refers at least to beers prepared from mashes prepared from malted cereals as well as mashes prepared from unmalted cereals, and mashes prepared from a mixture of malted and unmalted cereals.
  • beer also refers to beers prepared with adjuncts and beers with all possible alcohol contents.
  • reduced-alcohol beer herein refers to a beer with an alcohol content of between 1.2% to 4.2% alcohol by volume (ABV).
  • low-alcohol beer herein refers to a beer with an alcohol content of between 0.5% to 1.2% ABV.
  • non-alcohol beer refers to a beer with an alcohol content of less than 0.5% ABV.
  • hopped wort refers to a wort whereto hops have been added either prior to boiling or after boiling of the wort.
  • genetically modified organism herein has the meaning as defined by the 1996 Organic Trade Association and refers to an organism, such as a microorganism, such as yeast, which have been made with techniques that alter the molecular or cell biology of an organism by means that are not possible under natural conditions or processes.
  • non-genetically modified refers to a microorganism which does not contain heterologous genes.
  • mutant should be understood as a strain derived from a strain of the invention by means of e.g. genetic engineering, radiation and/or chemical treatment. It is preferred that the mutant is a functionally equivalent mutant, e.g. a mutant that has substantially the same, or improved, properties (e.g. regarding enhancement of desirable beer flavor compounds) as the mother strain. Such a mutant is a part of the present invention.
  • mutant refers to a strain obtained by subjecting a strain of the invention to any conventionally used mutagenization treatment including treatment with a chemical mutagen such as ethane methane sulphonate (EMS) or N-methyl-N′-nitro-N-nitroguanidine (NTG), UV light or to a spontaneously occurring mutant.
  • a mutant may have been subjected to several mutagenization treatments (a single treatment should be understood one mutagenization step followed by a screening/selection step), but it is presently preferred that no more than 20, or no more than 10, or no more than 5, treatments (or screening/selection steps) are carried out.
  • a presently preferred mutant less that 5%, or less than 1% or even less than 0.1% of the nucleotides in the yeast genome have been shifted with another nucleotide, or deleted, compared to the mother strain.
  • variant should be understood as a strain which is functionally equivalent to a strain of the invention, e.g. having substantially the same, or improved, properties (e.g. regarding enhancement of desirable beer flavor compounds).
  • malt is prepared from dried, germinated cereal grains (mainly barley or wheat) and grounded into a grist which may contain unmalted adjuncts.
  • the grist is mashed (mixed with water and steeped) to allow enzymes in the malt to convert the starch into sugars.
  • the grain particles and adjuncts are separated from the liquid wort in a process called lautering.
  • the malt making and mashing steps can be skipped by adding water to malt extract.
  • hops and/or other ingredients such as herbs and sugars
  • the wort is boiled (hops may also be added after boiling), cooled and aerated.
  • the wort is then moved to a fermentation tank and fermented by the addition of a brewer's yeast.
  • the primary fermentation lasting typically 5 to 10 days, may be followed by a secondary fermentation step using a further brewer's yeast.
  • Hops are added to the wort to balance the sweetness of the malt with bitterness and impart onto the beer desirable flavors and aromas.
  • Several varieties exist including but not limited to Ahtanum, Amarillo, Apollo, Cascade, Centennial, Chinook, Citra, Cluster, Columbus, Crystal, Eroica, Galena, Glacier, Greenburg, Horizon, Liberty, Millenium, Mount Hood, Mount Rainier, Newport, Nugget, Palisade, Santiam, Simcoe, Sterling, Summit, Tomahawk, Ultra, Vanguard, Warrior, Willamette, Zeus, Admiral, Brewer's Gold, Bullion, Challenger, First Gold, Fuggles, Goldings, Herald, Northdown, Northern Brewer, Phoenix, Pilot, Pioneer, Progress, Target, Whitbread Golding Variety (WGV), Hallertau, Hersbrucker, Saaz, Tettnang, Spelt, Feux-Coeur Francais, Galaxy, Green Bullet, Motueka, Nelson Sauvin, Pacific Gem, Pacific Jade,
  • Pichia spp. yeast strains when combined with the addition of different varieties of hops to the wort, have advantageous properties useful in the fermentation process of beer brewing.
  • the Pichia spp. yeast strains enhance desirable flavors from the hops when the hopped wort is fermented with the Pichia spp. yeast strains in sequence to fermentation of the hopped wort with a yeast strain conventionally used for beer brewing, including yeasts of the genera Saccharomyces and Brettanomyces.
  • the method for brewing beer of the present invention comprises the steps of:
  • step c) and step d) are carried out sequentially, i.e. the hopped wort is inoculated first with a Pichia spp. yeast strain under conditions suitable for fermentation and subsequently the hopped wort is inoculated with a second yeast strain under conditions suitable for fermentation.
  • the fermentation in step c) is allowed to proceed for at least 12 hours, such as for at least 24 hours, such as for at least 36 hours, such as for at least 48 hours, such as for at least 60 hours, such as at least 72 hours prior to inoculation of the second yeast strain in step d). In a most preferred embodiment the fermentation in step c) is allowed to proceed for at least 36 hours.
  • the at least one variety of hop may be selected from the list consisting of Ahtanum, Amarillo, Apollo, Cascade, Centennial, Chinook, Citra, Cluster, Columbus, Crystal, Eroica, Galena, Glacier, Greenburg, Horizon, Liberty, Millenium, Mount Hood, Mount Rainier, Newport, Nugget, Palisade, Santiam, Simcoe, Sterling, Summit, Tomahawk, Ultra, Vanguard, Warrior, Willamette, Zeus, Admiral, Brewer's Gold, Bullion, Challenger, First Gold, Fuggles, Goldings, Herald, Northdown, Northern Brewer, Phoenix, Pilot, Pioneer, Progress, Target, Whitbread Golding Variety (WGV), Hallertau, Hersbrucker, Saaz, Tettnang, Spalt, Feux-Coeur Francais, Galaxy, Green Bullet, Motueka, Nelson Sauvin, Pacific Gem, Pacific Jade, Pacifica
  • the at least one hop variety is selected from the group consisting of Saaz, Nelson Sauvin, Cascade and Amarillo.
  • the at least one variety of hop may also be selected from the list consisting of Bravo, Calypso, Chelan, Comet, El Dorado, San Juan Ruby Red, Satus, Sonnet Golding, Super Galena, Tillicum, Bramling Cross, Pilgrim.
  • the fermentation in step c) is carried out at a temperature of between about 12° C. and about 28° C. In a more preferred embodiment the fermentation temperature in step c) is between about 20° C. and about 22° C. Most preferably the fermentation temperature in step c) is about 20° C.
  • the Pichia spp. yeast strain is left in the fermentation culture during fermentation with the second yeast strain.
  • the Pichia spp. yeast strain may also be removed prior to inoculation of the second yeast strain by techniques known to the skilled person.
  • step c) and step d) are carried out at the same time by co-inoculation of the first yeast strain of step c) and the second yeast strain of step d).
  • the second fermentation step in step d) is allowed to proceed for at least 12 hours, such as at least 24 hours, such as at least 48 hours, such as at least 72 hours, such as at least 96 hours, such as at least 120 hours.
  • the Pichia spp. yeast strain is a non-genetically modified yeast strain.
  • the Pichia spp. yeast strain is a Pichia kluyveri yeast strain.
  • the Pichia kluyveri yeast strain is selected from the group consisting of the Pichia kluyveri PK-KR1 and PK-KR2 strains as deposited on 24 Aug. 2006 at the National Measurement Institute, 541-65 Clarke Street, South Melbourne, Victoria 3205, Australia, by University of Auckland, School of Biological Sciences, Auckland 1142, New Zealand, and given the accession numbers V06/022711 and V06/022712, respectively, and mutants and variants thereof.
  • the strains are described in international patent application WO 2009/110807.
  • the second yeast strain is selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces pastorianus and Brettanomyces bruxellensis .
  • the second yeast strain is a Saccharomyces cerevisiae yeast strain.
  • the beer is a low-alcohol, reduced-alcohol or non-alcohol beer.
  • the use of a Pichia spp. yeast strain in step c) increases the content of at least one flavor compound selected from the group consisting of isoamyl acetate, isobutyl acetate, ethyl propionate, ethyl valerate, ethyl octanoate and ethyl decanoate by at least 50%, such as by at least 100%, such as by at least 150%, such as by at least 200%, such as by at least 250%, such as by at least 300%.
  • the use of a Pichia spp. yeast strain in step c) increases the content of at least one flavor compound selected from the group consisting of isoamyl acetate, isobutyl acetate, ethyl propionate and ethyl valerate by at least 50%, such as by at least 100%, such as by at least 150%, such as by at least 200%, such as by at least 250%, such as by at least 300%.
  • the use a Pichia spp. yeast strain in step c) increases the content of at least two or more flavor compounds selected from the group consisting of isoamyl acetate, isobutyl acetate, ethyl propionate, ethyl valerate, ethyl octanoate and ethyl decanoate by at least 50%, such as by at least 100%, such as by at least 150%, such as by at least 200%, such as by at least 250%, such as by at least 300%.
  • the use of a Pichia spp. yeast strain in step c) increases the content of at least two or more flavor compounds selected from the group consisting of isoamyl acetate, isobutyl acetate, ethyl propionate and ethyl valerate by at least 50%, such as by at least 100%, such as by at least 150%, such as by at least 200%, such as by at least 250%, such as by at least 300%.
  • step c) increases the content of the thiol 3-mercaptohexyl acetate by at least 10%, such as by at least 20%, such as by at least 30%, such as by at least 40%, such as by at least 50%.
  • step b) when the at least one hop variety in step b) is Saaz and the fermentation in step c) is carried out at a temperature of about 20° C. the use of the Pichia spp. yeast strain in step c) increases the level of ethyl decanoate with at least 50% more than when the at least one hop variety is Cascade, Nelson Sauvin or Amarillo.
  • step b) when the at least one hop variety in step b) is Nelson Sauvin and the fermentation in step c) is carried out at a temperature of about 20° C. the use of the Pichia spp. yeast strain in step c) increases the level of ethyl decanoate with at least 50% more than when the at least one hop variety is Cascade or Amarillo.
  • step b) when the at least one hop variety in step b) is Cascade or Nelson Sauvin and the fermentation in step c) is carried out at a temperature of about 20° C. the use of the Pichia spp. yeast strain in step c) increases the level of ethyl valerate with at least 50% more than when the at least one hop variety is Amarillo and Saaz.
  • step b) when the at least one hop variety in step b) is Cascade and the fermentation in step c) is carried out at a temperature of about 20° C. the use of the Pichia yeast strain in step c) increases the level of ethyl hexanoate and ethyl octanoate with at least 50% more than when the at least one hop variety is Nelson Sauvin, Amarillo or Saaz.
  • the at least one hop variety in step b) is any variety selected from the group consisting of Ahtanum, Amarillo, Apollo, Cascade, Centennial, Chinook, Citra, Cluster, Columbus, Crystal, Eroica, Galena, Glacier, Greenburg, Horizon, Liberty, Millenium, Mount Hood, Mount Rainier, Newport, Nugget, Palisade, Santiam, Simcoe, Sterling, Summit, Tomahawk, Ultra, Vanguard, Warrior, Willamette, Zeus, Admiral, Brewer's Gold, Bullion, Challenger, First Gold, Fuggles, Goldings, Herald, Northdown, Northern Brewer, Phoenix, Pilot, Pioneer, Progress, Target, Whitbread Golding Variety (WGV), Hallertau, Hersbrucker, Saaz, Tettnang, Spalt, Feux-Coeur Francais, Galaxy, Green Bullet, Motueka, Nelson Sauvin, Pacific Gem, Pacific Jade, Pacifica, Pride of Ringwood, Riwaka, Southern Cross
  • yeast strain in step c) will result in a flavor profile of levels of the flavor compounds isoamyl acetate, isobutyl acetate, ethyl propionate and ethyl valerate which is different from a flavor profile resulting from use of the Pichia spp.
  • the at least one hop variety in step b) is any variety selected from the group consisting of Ahtanum, Amarillo, Apollo, Cascade, Centennial, Chinook, Citra, Cluster, Columbus, Crystal, Eroica, Galena, Glacier, Greenburg, Horizon, Liberty, Millenium, Mount Hood, Mount Rainier, Newport, Nugget, Palisade, Santiam, Simcoe, Sterling, Summit, Tomahawk, Ultra, Vanguard, Warrior, Willamette, Zeus, Admiral, Brewer's Gold, Bullion, Challenger, First Gold, Fuggles, Goldings, Herald, Northdown, Northern Brewer, Phoenix, Pilot, Pioneer, Progress, Target, Whitbread Golding Variety (WGV), Hallertau, Hersbrucker, Saaz, Tettnang, Spalt, Feux-Coeur Francais, Galaxy, Green Bullet, Motueka, Nelson Sauvin, Pacific Gem, Pacific Jade, Pacifica, Pride of Ringwood, Riwaka, Southern
  • yeast strain in step c) will result in a flavor profile of levels of the flavor compounds isoamyl acetate, isobutyl acetate, ethyl propionate and ethyl valerate which is different from a flavor profile resulting from use of the Pichia spp.
  • the present invention in a second aspect relates to a beer obtainable by the method of the first aspect.
  • the beer according to the present invention may comprise detectable amounts of Pichia spp. yeast. Although, in some instances the yeast is removed from the beer after fermentation.
  • the beer brewed with Pichia spp. contains a different flavor profile when different hop varieties are used.
  • the ethyl decanoate level is at least 50% more increased when Saaz hop is used, compared to when Cascade, Nelson Sauvin or Amarillo is used and is at least 50% increased when Nelson Sauvin is used, compared to when Cascade or Amarillo is used in the fermentations performed at 20° C. (see FIG. 21 ).
  • the ethyl valerate concentration is at least 50% increased when Cascade or Nelson Sauvin hop is used, compared to when Amarillo or Saaz is used in fermentations performed at 20° C. (see FIG. 15 ).
  • the ethyl hexanoate and ethyl octanoate concentrations are at least increased with 15% when Cascade hops is used, compared to when Nelson Sauvin, Amarillo or Saaz hops are used in fermentations performed at 20° C. (see FIGS. 17 and 19 ).
  • the beer brewed using Pichia spp. will contain detectable amounts of thiols (in particular 3-mercaptohexyl acetate (3MHA) and 3-mercaptohexan-1-ol (3MH)) such as described for wine in WO 2009/110807.
  • thiols in particular 3-mercaptohexyl acetate (3MHA) and 3-mercaptohexan-1-ol (3MH)
  • Per hop variety 4 fermentations were carried out. Two fermentation temperatures were used: 20° C. and 22° C. Per fermentation temperature, 2 different inoculations were done: one bottle was inoculated with Pichia kluyveri PK-KR1 (for sequential inoculation) and the second bottle was inoculated with a commercial Saccharomyces cerevisiae wheat yeast strain, Safbrew WB-06 (Lesaffre) (control fermentation) according to the supplier recommendations. Both yeast strains were inoculated at 5 million cells per ml. Table 1 gives an overview of the fermentation set-up.
  • the wheat yeast strain (Safbrew WB-06) was inoculated after 2 days to the Pichia kluyveri inoculated ferments to complete the fermentation. All fermentations were ended after 13 days. Samples were taken at day 5 and day 13.
  • GC-FID flame ionisation detection
  • Ethanol was measured with the Ethanol Enzymatic Bioanalysis kit of Boehringer Mannheim.
  • FIGS. 1 and 2 aldehydes
  • FIG. 3-10 acetate esters
  • FIG. 11-16 short-chain ethyl esters
  • FIG. 17-22 medium-chain ethyl esters
  • FIG. 23-26 higher alcohols
  • Ethanol concentrations were measured with a kit (see Materials and Methods). The results of the ethanol concentrations are shown in Table 2.
  • Acetaldehyde concentrations were always higher in the sequential inoculation, compared to the control fermentation (see FIG. 1-2 ). However, at the end of fermentation, similar values are obtained for both the sequential and control fermentation. As brewers don't want too high concentrations of this compound in the beer, the effect of the sequential inoculation is negligible.
  • Ethyl acetate which is a negative compound at high concentrations, but easily masked by other acetate esters, was increased as well in the sequential fermentations, but only two times the amount, compared to the controls.
  • the fermentation with Cascade hop produced the less ethyl acetate of all the fermentations.
  • Hexyl acetate increased only a little bit in all the sequential fermentations, compared to the control fermentations.
  • the higher alcohols are the only compounds of which the concentrations did not increase significantly in the sequential fermentations, compared to the control fermentations.
  • the control beer and Pichia beer were made on a 15 HL scale in a brewery.
  • the wort consisted of malt varieties typical for a pale ale beer, and the hop variety used was Cascade. 15 hl of wort was used to inoculate the Pichia kluyveri PK-KR1. Inoculation rate was calculated to be between 1-2 million cells per ml. Every day, a sample was taken for measurement of cell counts (see Table 3) and flavor analysis was performed on the final beer.
  • the normal brewing yeast was added (London Ale yeast from White Labs).
  • the yeast was already used for making one beer and should therefore be optimal for this brew (2nd generation).
  • the yeast was added while flushing air through the brew. Therefore, it was possible to have an idea of the smell of the brew while the brewing yeast was added.
  • the smell was very fruity (banana, pineapple-like).
  • Fermentation was carried out until the sugar had been completely assimilated by the yeast. After that, the green beer was matured at 7-8° C. for at least a week.
  • the fermentation was completed and the fermentation temperature was lowered to 8° C.
  • the beer matured for 7 days with the yeast still in the fermentor.
  • the yeast was taken out and the temperature was decreased to 2° C.
  • the beer was filtered, pasteurized and put into bottles.
  • GC-FID flame ionization detection
  • the flavor analysis clearly shows that the beer fermented with the Pichia kluyveri has increased concentrations of flavor compounds.
  • the esters isoamyl acetate ( FIG. 29 ), ethyl propionate ( FIG. 30 ), ethyl valerate( FIG. 32 ), ethyl octanoate ( FIG. 34 ) and ethyl decanoate ( FIG. 35 ) are increased in high amounts. These esters are responsible for the fruity flavors in beers and are therefore of major importance.
  • the Pichia kluyveri PK-KR1 and PK-KR2 strains were deposited on 24 Aug. 2006 at the National Measurement Institute, 51-65 Clarke Street, South Melbourne, Victoria 3205, Australia, by University of Auckland, School of Biological Sciences, Auckland 1142, New Zealand, and given the accession numbers V06/022711 and V06/022712, respectively, as described in WO 2009/110807.
  • Domizio P Romani C, Lencioni L, Comitini F, Gobbi M, Mannazzu I, and Ciani M. (2011) Outlining a future for non- Saccharomyces yeasts: selection of putative spoilage wine strains to be used in combination with Saccharomyces cerevisiae for grape juice fermentation. Int J Food Microbiol. 147:170-80.
  • Verachtert H Kumara H M C, Dawoud E (1989) Yeast in mixed cultures with emphasis on Iambic beer brewing.
  • Verachtert H De Mot R (eds) Yeast—biotechnology and biocatalysis. Dekker, New York, pp 429-478.
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