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WO2007144393A1 - Procédé de brassage - Google Patents

Procédé de brassage Download PDF

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Publication number
WO2007144393A1
WO2007144393A1 PCT/EP2007/055863 EP2007055863W WO2007144393A1 WO 2007144393 A1 WO2007144393 A1 WO 2007144393A1 EP 2007055863 W EP2007055863 W EP 2007055863W WO 2007144393 A1 WO2007144393 A1 WO 2007144393A1
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WO
WIPO (PCT)
Prior art keywords
acid sequence
process according
beer
wort
pullulanase
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/EP2007/055863
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English (en)
Inventor
Niels Elvig
Per Linaa Joergensen
Michael Thomas
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Novozymes AS
Novozymes Inc
Original Assignee
Novozymes AS
Novozymes Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Novozymes AS, Novozymes Inc filed Critical Novozymes AS
Publication of WO2007144393A1 publication Critical patent/WO2007144393A1/fr
Priority to US12/326,326 priority Critical patent/US8765199B2/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • 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
    • C12C7/04—Preparation or treatment of the mash
    • 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
    • C12C5/00—Other raw materials for the preparation of beer
    • C12C5/004—Enzymes

Definitions

  • the present invention relates to an improved mashing process for production of a brewer's wort and for production of a beer.
  • enzymes are often added as a supplement when mashing malt is low in enzymes or to allow use of all adjunct grists. Enzymes may also be applied in mashing of well modified malts with high enzyme content in order to increase the extract recovery as well as the amount of fermentable sugars. It is thus well known to apply debranching enzymes, e.g. isoamylase or pullulanase to increase the yield fermentable sugars. Debranching enzymes may be applied in processes for production of low calorie beer. Such processes are the subject of
  • the present inventors have now surprisingly discovered that by using a certain pullulanase mashing, can be achieved using a smaller amount of enzyme protein.
  • the invention provides a process for producing a brewers wort comprising forming a mash from a grist, and contacting said mash with a pullulanase (E. C. 3.2.1.41 ), wherein said pullulanase has an amino acid sequence which a) is at least 50% identical to the amino acid sequence shown in SEQ ID NO:3, or b) is encoded by a nucleic acid sequence which hybridizes under low stringency conditions with i) a complementary strand of a nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO:3, or ii) a subsequence of (i) of at least 100 nucleotides.
  • a pullulanase E. C. 3.2.1.41
  • the invention provides a wort produced by the process of the first aspect.
  • the invention provides concentrated and/or dried wort produced by the process of the first aspect.
  • the invention provides beer produced from the wort of the second and third aspect.
  • a composition suitable for use in the process of the first aspect comprising pullulanase (E. C. 3.2.1.41 ), glucoamylase and optionally alpha-amylase, wherein the pullulanase has an amino acid sequence which a) is at least 50% identical to the amino acid sequence shown in SEQ ID NO:3, or b) is encoded by a nucleic acid sequence which hybridizes under low stringency conditions with i) a complementary strand of a nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO:3, or ii) a subsequence of (i) of at least 100 nucleotides.
  • Brewing processes are well-known in the art, and generally involve the steps of malting, mashing, and fermentation.
  • Mashing is the process of converting starch from the milled barley malt and solid adjuncts into fermentable and unfermentable sugars to produce wort of the desired composition.
  • Traditional mashing involves mixing milled barley malt and adjuncts with water at a set temperature and volume to continue the biochemical changes initiated during the malting process.
  • the mashing process is conducted over a period of time at various temperatures in order to activate the endogenous enzymes responsible for the degradation of proteins and carbohydrates.
  • the principal enzymes responsible for starch conversion in a traditional mashing process are alpha- and beta-amylases.
  • Alpha-amylase very rapidly reduces insoluble and soluble starch by splitting starch molecules into many shorter chains that can be attacked by beta-amylase.
  • the disaccharide produced is maltose.
  • short branched glucose oligomers are produced.
  • the short branched glucose oligomers are non fermentable sugars and add to the taste as well as the calories of the finished beer.
  • the wort liquid extract
  • the solids solids
  • wort separation lautering, is important because the solids contain large amounts of protein, poorly modified starch, fatty material, silicates, and polyphenols (tannins).
  • the wort may be fermented with brewers yeast to produce a beer.
  • the short branched glucose oligomers formed during mashing may be further hydrolyzed by addition of exogenous enzymes (enzymes added in addition to the malt).
  • deranching enzymes such as pullulanase and isoamylase hydrolyses the branching alpha-1-6 glucosidic bonds in these oligomers, thereby releasing glucose or maltose and straight-chained oligomers which are subject to the action of endogenous (malt derived) and/or exogenous enzymes, e.g. alpha-amylases, beta-amylases and glucoamylases.
  • the present invention provides a new process suitable for producing a wort that is low in non- fermentable sugars.
  • the process applies an expressly selected pullulanase activity.
  • the term "grist” is understood as the starch or sugar containing material that's the basis for beer production, e.g. the barley malt and the adjunct. Generally, the grist is does not contain any added water.
  • malt is understood as any malted cereal grain, in particular barley.
  • adjunct is understood as the part of the grist which is not barley malt.
  • the adjunct may comprise any starch rich plant material, e.g. unmalted grain, such as barley, rice, corn, wheat, rye, sorghum and readily fermentable sugar and/or syrup.
  • mash is understood as a starch containing slurry comprising grist steeped in water.
  • wort is understood as the unfermented liquor run-off following extracting the grist during mashing.
  • sucrose is understood as the drained solids remaining when the grist has been extracted and the wort separated.
  • fermented wort i.e. an alcoholic beverage brewed from barley malt, optionally adjunct and hops.
  • homologous sequence is used to characterize a sequence having an amino acid sequence that is at least 70%, preferably at least 75%, or at least 80%, or at least 85%, or 90%, or at least 95%, at least 96%, at least 97%, at least 98% at least 99% or even at least 100% identical to a known sequence.
  • the relevant part of the amino acid sequence for the homology determination is the mature polypeptide, i.e., without the signal peptide.
  • homologous sequence is also used to characterize DNA sequences which hybridize at low stringency, medium stringency, medium/high stringency, high stringency, or even very high stringency with a known sequence.
  • Suitable experimental conditions for determining hybridization at low, medium, or high stringency between a nucleotide probe and a homologous DNA or RNA sequence involves presoaking of the filter containing the DNA fragments or RNA to hybridize in 5 x SSC (Sodium chloride/Sodium citrate, Sambrook et al. 1989) for 10 min, and prehybridization of the filter in a solution of 5 x SSC, 5 x Denhardt's solution (Sambrook et al. 1989), 0.5% SDS and 100 micrograms/ml of denatured sonicated salmon sperm DNA (Sambrook et al.
  • 5 x SSC Sodium chloride/Sodium citrate, Sambrook et al. 1989
  • identity when used about polypeptide or DNA sequences and referred to in this disclosure is understood as the degree of identity between two sequences indicating a derivation of the first sequence from the second.
  • the identity may suitably be determined by means of computer programs known in the art such as GAP provided in the GCG program package (Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wisconsin, USA 5371 1 ) (Needleman, S. B. and Wunsch, CD., (1970), Journal of Molecular Biology, 48, 443-453.
  • the following settings for polypeptide sequence comparison are used: GAP creation penalty of 3.0 and GAP extension penalty of 0.1.
  • the degree of identity between an amino acid sequence of the present invention and a different amino acid sequence is calculated as the number of exact matches in an alignment of the two sequences, divided by the length of the "invention sequence” or the length of the "foreign sequence", whichever is the shortest. The result is expressed in percent identity.
  • the invention provides a process for producing a brewers wort comprising forming a mash from a grist, and contacting said mash with a pullulanase (E. C. 3.2.1.41 ), wherein said pullulanase has an amino acid sequence which a) is at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, or at least 85%, or 90%, or at least 95%, at least 96%, at least 97%, at least 98% or even at least 99% identical to the amino acid sequence shown in SEQ ID NO:3, or b) is encoded by a nucleic acid sequence which hybridizes under low stringency, medium stringency, medium/high stringency, high stringency, or even very high stringency with i) a complementary strand of a nucleic acid sequence encoding the amino acid sequence shown in SEQ ID NO:3, or ii) a subsequence of (i) of at least 100 nucleot
  • the pullulanase has an amino acid sequence which differs by no more than 100 amino acids, preferably by no more than 80 amino acids, more preferred by no more than 50 amino acids, more preferably by no more than 30 amino acids, even more preferably by no more than 20 amino acids, and most preferably by no more than 10 amino acids from the amino acid sequence of SEQ ID NO: 3.
  • the grist of the first aspect comprises starch containing malted grain and/or adjunct.
  • the grist may preferably comprises from 0% to 100%, preferably from 20% to 1000%, preferably from 30% to 100%, more preferably from 40% to 100%, even more preferably from 50% to 100%, yet more preferably from 100% to 80%, most preferably from 100% to 80% adjunct, or even most preferably from 90% to 100% adjunct, unmalted grain and/or unmalted barley.
  • the adjunct is composed of 100% unmalted barley.
  • the grist preferably comprises from 0% to 100%, preferably from 20% to 100%, preferably from 30% to 100%, more preferably from 40% to 100%, even more preferably from 50% to 100%, yet more preferably from 60% to 100%, or most preferably from 70% to 100%, or even most preferably from 90% to 100% malted grain and/or malted barley.
  • the grist comprises approximately 50% malted grain, e.g. malted barley, and approximately 50% adjunct, e.g. unmalted grain, such as unmalted barley.
  • Malted grain used in the process of the first aspect may comprise any malted grain, and preferably malted grain selected from malted barley, wheat, rye, sorghum, millet, corn, and rice, and most preferably malted barley.
  • the adjunct used in the process of the first aspect may be obtained from tubers, roots, stems, leaves, legumes, cereals and/or whole grain.
  • the adjunct may comprise raw and/or refined starch and/or sugar containing material derived from plants like wheat, rye, oat, corn, rice, milo, millet, sorghum, potato, sweet potato, cassava, tapioca, sago, banana, sugar beet and/or sugar cane.
  • the adjunct comprises unmalted grain, e.g. unmalted grain selected from the list consisting of barley, wheat, rye, sorghum, millet, corn, and rice, and most preferably unmalted barley.
  • Adjunct comprising readily fermentable carbohydrates such as sugars or syrups may be added to the barley malt mash before, during or after mashing process of the invention but is preferably added after the mashing process.
  • a pullulanase (E. C. 3.2.1.41 ) enzyme activity is exogenously supplied and present in the mash.
  • the pullulanase may be added to the mash ingredients, e.g. the water and/or the grist before, during or after forming the mash.
  • an alpha-amylase (E. C. 3.2.1.1 ) and/or a glucoamylase (E. C. 3.2.1.3) is added and present in the mash together with the pullulanase.
  • a further enzyme is added to the mash, said enzyme being selected from the group consisting of isoamylase, protease, laccase, xylanase, lipase, phospholipolase, phytase, phytin and esterase.
  • starch extracted from the grist is gradually hydrolyzed into fermentable sugars and smaller dextrins.
  • the mash is starch negative to iodine testing, before extracting the wort.
  • the mashing process generally apply a controlled stepwise increase in temperature, where each step favors one enzymatic action over the other, eventually degrading proteins, cell walls and starch.
  • Mashing temperature profiles are generally known in the art.
  • the saccharification (starch degradation) step in the mashing process is preferably performed between 60 0 C and 66 ° C, more preferably between 61 0 C and 65 0 C, even more preferably between 62 0 C and 64 0 C, and most preferably between 63 0 C and 64 0 C.
  • the saccharification temperature is 64 0 C.
  • Obtaining the wort from the mash typically includes straining the wort from the spent grains, i.e. the insoluble grain and husk material forming part of grist. Hot water may be run through the spent grains to rinse out, or sparge, any remaining extract from the grist.
  • the application of a thermostable cellulase in the process of the present invention results in efficient reduction of beta- glucan level facilitating wort straining thus ensuring reduced cycle time and high extract recovery.
  • the extract recovery is at least 80%, preferably at least 81 %, more preferably at least 82%, even more preferably at least 83%, such as at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, and most preferably at least 91 %.
  • the wort may be used as it is or it may be dewatered to provide a concentrated and/or dried wort e.g.
  • the concentrated and/or dried wort may be used as brewing extract, as malt extract flavoring, for non-alcoholic malt beverages, malt vinegar, breakfast cereals, for confectionary etc.
  • the wort is fermented to produce an alcoholic beverage, preferably a beer, e.g., ale, strong ale, bitter, stout, porter, lager, export beer, malt liquor, barley wine, happoushu, high-alcohol beer, low-alcohol beer, low-calorie beer or light beer.
  • Fermentation of the wort may include pitching the wort with a yeast slurry comprising fresh yeast, i.e. yeast not previously used for the invention or the yeast may be recycled yeast.
  • the yeast applied may be any yeast suitable for beer brewing, especially yeasts selected from Saccharomyces spp. such as S. cerevisiae and S. uvarum, including natural or artificially produced variants of these organisms.
  • the methods for fermentation of wort for production of beer are well known to the person skilled in the arts.
  • the process of the invention may include adding silica hydrogel to the fermented wort to increase the colloidal stability of the beer.
  • the processes may further include adding kieselguhr to the fermented wort and filtering to render the beer bright.
  • beer produced from the wort of the second or third aspect such as a beer produced by fermenting the wort to produce a beer.
  • the beer may be any type of beer, e.g., ales, strong ales, stouts, porters, lagers, bitters, export beers, malt liquors, happoushu, high-alcohol beer, low-alcohol beer, low-calorie beer or light beer.
  • the enzymes to be applied in the present invention should be selected for their ability to retain sufficient activity at the process temperature of the processes of the invention, as well as under the pH regime in the mash and should be added in effective amounts.
  • the enzymes may be derived from any source, preferably from a plant or an alga, and more preferably from a microorganism, such as from a bacterium or a fungus.
  • a preferred pullulanase enzyme to be used in the processes and/or compositions of the invention is a pullulanase having an amino acid sequence which is at least 50%, preferably 60% at least, more preferably at least 70%, even more preferably at least 80%, such as at least 90%, at least 95%, at least 98% or even 100% identical to the sequence shown in SEQ ID NO:3.
  • Most preferably the pullulanase is derived from Bacillus acidopullulyticus.
  • the pullulanase may have the amino acid sequence disclosed by Kelly et al., 1994 (FEMS Microbiol. Letters, 115, 97-106) or a homologous sequence.
  • Isoamylase (E.C. 3.2.1.68) Another enzyme applied in the processes and/or compositions of the invention may be an alternative debranching enzyme, such as an isoamylase (E.C. 3.2.1.68).
  • lsoamylase hydrolyses alpha-1 ,6-D-glucosidic branch linkages in amylopectin and beta-limit dextrins and can be distinguished from pullulanases by the inability of isoamylase to attack pullulan, and by the limited action on alpha-limit dextrins.
  • Isoamylase may be added in effective amounts well known to the person skilled in the art. Isoamylase may be added alone or together with a pullulanase.
  • Alpha-amylase (EC 3.2.1.1 )
  • a particular alpha-amylase enzyme to be used in the processes and/or compositions of the invention may be a Bacillus alpha-amylase.
  • Bacillus alpha-amylases include alpha- amylase derived from a strain of B. licheniformis, B. amyloliquefaciens, and B. stearothermophilus.
  • a contemplated Bacillus alpha-amylase is an alpha-amylase as defined in WO99/19467 on page 3, line 18 to page 6, line 27.
  • a preferred alpha-amylase has an amino acid sequence having at least 90% identity to SEQ ID NO:4 in WO99/19467, such as at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or particularly at least 99%.
  • Most preferred variants of the maltogenic alpha-amylase comprise the variants disclosed in WO99/43794.
  • Contemplated variants and hybrids are described in WO96/23874, WO97/41213, and WO99/19467.
  • an alpha-amylase (E. C. 3.2.1.1 ) from B. stearothermophilus having the amino acid sequence disclosed as SEQ ID NO:4 in WO99/19467 with the mutations: 1181 * + G182 * + N193F.
  • Bacillus alpha-amylases may be added in the amounts of 1.0-1000 NU/kg DS, preferably from 2.0-500 NU/kg DS, preferably 10-200 NU/kg DS.
  • Another particular alpha-amylase to be used in the processes of the invention may be any fungal alpha-amylase, e.g. an alpha-amylase derived from a species within Aspergillus, and preferably from a strain of Aspergillus niger.
  • fungal alpha-amylases which exhibit a high identity, i.e. at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or even at least 90% identity to the amino acid sequences shown SEQ ID NO:1 in WO 2002/038787.
  • Fungal alpha-amylases may be added in an amount of 1-1000 AFAU/kg DS, preferably from 2-500 AFAU/kg DS, preferably 20-100 AFAU/kg DS.
  • a further particular enzyme to be used in the processes and/or compositions of the invention may be a glucoamylase (E.C.3.2.1.3) derived from a microorganism or a plant.
  • glucoamylases of fungal or bacterial origin selected from the group consisting of Aspergillus glucoamylases, in particular A. niger GI or G2 glucoamylase (Boel et al. (1984), EMBO J. 3 (5), p.
  • glucoamylases include Talaromyces glucoamylases, in particular derived from Talaromyces emersonii (WO99/28448), Talaromyces leycettanus (US patent no. Re. 32,153), Talaromyces duponti, Talaromyces thermophilus (US patent no. 4,587,215).
  • Preferred glucoamylases include the glucoamylases derived from Aspergillus oryzae, such as a glucoamylase having at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or particularly at least 99% or even at least 90% identity to the amino acid sequence shown in SEQ ID NO:2 in WO00/04136.
  • glucoamylases include the glucoamylases derived from Talaromyces emersonii such as a glucoamylase having at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, or particularly at least 99% or even at least 90% identity to the amino acid sequence shown in SEQ ID NO:2 in 60/738,448
  • Bacterial glucoamylases contemplated include glucoamylases from the genus Clostridium, in particular C. thermoamylolyticum (EP 135,138), and C. thermohydrosulfuricum (WO86/01831 ).
  • Glucoamylases may be added in effective amounts well known to the person skilled in the art.
  • Suitable proteases include microbial proteases, such as fungal and bacterial proteases.
  • Preferred proteases are acidic proteases, i.e., proteases characterized by the ability to hydrolyze proteins under acidic conditions below pH 7.
  • proteases are responsible for reducing the overall length of high-molecular-weight proteins to low-molecular-weight proteins in the mash.
  • the low-molecular-weight proteins are a necessity for yeast nutrition and the high-molecular-weight-proteins ensure foam stability.
  • protease should be added in a balanced amount which at the same time allows amble free amino acids for the yeast and leaves enough high-molecular- weight-proteins to stabilize the foam.
  • Proteases may be added in the amounts of 0.1-1000 AU/kg DS, preferably 1-100 AU/kg DS and most preferably 5-25 AU/kg DS.
  • the cellulase may be of microbial origin, such as derivable from a strain of a filamentous fungus (e.g., Aspergillus, Trichoderma, Humicola, Fusarium).
  • a filamentous fungus e.g., Aspergillus, Trichoderma, Humicola, Fusarium.
  • Specific examples of cellulases include the endo-glucanase (endo-glucanase I) obtainable from H. insolens and further defined by the amino acid sequence of fig. 14 in WO 91/17244 and the 43 kD H. insolens endo-glucanase described in WO 91/17243.
  • a particular cellulase to be used in the processes of the invention may be an endo- glucanase, such as an endo-1 ,4-beta-glucanase.
  • an endo-glucanase such as an endo-1 ,4-beta-glucanase.
  • Commercially available cellulase preparations which may be used include CELLUCLAST®, CELLUZYME®, CEREFLO® and ULTRAFLO® (available from Novozymes A/S), LAMINEXTM and SPEZYME® CP (available from Genencor Int.) and ROHAMENT® 7069 W (available from Rohm, Germany).
  • Beta-glucanases may be added in the amounts of 1.0-10000 BGU/kg DS, preferably from 10-5000 BGU/kg DS, preferably from 50-1000 BGU/kg DS and most preferably from 100-500 BGU/kg DS.
  • Pullulanase 1 derived from Bacillus acidopullulyticus and having the sequence showed in SEQ ID NO:1. Pullulanase 1 is available from Novozymes as Promozyme 400L.
  • Pullulanase 2 derived from Bacillus deramificans (US Patent 5,736375) and having the sequence showed in SEQ ID NO:2.
  • Pullulanase 2 is available from Novozymes as Promozyme D2.
  • Pullulanase 3 derived from Bacillus acidopullulyticus and having the sequence showed in SEQ ID N0:3.
  • NU Alpha-amylase activity
  • Alpha-amylase activity may be determined using potato starch as substrate. This method is based on the break-down of modified potato starch by the enzyme, and the reaction is followed by mixing samples of the starch/enzyme solution with an iodine solution. Initially, a blackish-blue color is formed, but during the break-down of the starch the blue color gets weaker and gradually turns into a reddish-brown, which is compared to a colored glass standard.
  • KNU One Kilo Novo alpha amylase Unit
  • One KNU is defined as the amount of enzyme which, under standard conditions (i.e. at 37°C +/- 0.05; 0.0003 M Ca2+; and pH 5.6) degrades 5.26 g starch dry matter (Merck Amylum solubile).
  • AFAU Acid alpha-amylase activity
  • Acid alpha-amylase activity may be measured in AFAU (Acid Fungal Alpha-amylase Units), which are determined relative to an enzyme standard. 1 FAU is defined as the amount of enzyme which degrades 5.260 mg starch dry matter per hour under the below mentioned standard conditions.
  • Acid alpha-amylase an endo-alpha-amylase (1 ,4-alpha-D-glucan-glucanohydrolase, E. C. 3.2.1.1 ) hydrolyzes alpha-1 ,4-glucosidic bonds in the inner regions of the starch molecule to form dextrins and oligosaccharides with different chain lengths.
  • the intensity of color formed with iodine is directly proportional to the concentration of starch.
  • Amylase activity is determined using reverse colorimetry as a reduction in the concentration of starch under the specified analytical conditions.
  • Iodine (I2) 0.03 g/L
  • AGU The Novo Glucoamylase Unit (AGU) is defined as the amount of enzyme, which hydrolyzes 1 micromole maltose per minute at 37°C and pH 4.3.
  • the activity is determined as AGU/ml by a method modified after (AEL-SM-0131 , available on request from Novozymes) using the Glucose GOD-Perid kit from Boehringer Mannheim, 124036. Standard: AMG-standard, batch 7-1195, 195 AGU/ml. 375 microL substrate (1 % maltose in 50 mM Sodium acetate, pH 4.3) is incubated 5 minutes at 37°C. 25 microL enzyme diluted in sodium acetate is added. The reaction is stopped after 10 minutes by adding 100 microL 0.25 M NaOH. 20 microL is transferred to a 96 well microtitre plate and 200 microL GOD-Perid solution (124036, Boehringer Mannheim) is added. After 30 minutes at room temperature, the absorbance is measured at 650 nm and the activity calculated in AGU/ml from the AMG-standard. A detailed description of the analytical method (AEL-SM-0131 ) is available on request from Novozymes.
  • Enzymes were added at 0 minutes. Glucoamylase and alpha-amylase were added to all treatments in amounts of 1000 AGU/kg DS and 250 AFAU/kg DS respectively. Pullulanase was added according to table 1. The samples were boiled 10 minutes and filtered (Pore size 0.20 ⁇ m). The samples were analyzed by HPLC and % non fermentable carbohydrate (DP4/4+) was calculated.
  • Pullulanase 3 is the most efficient enzyme. Consequently, less Pullulanase 3 enzyme protein is needed to reduce the amount of non-fermentable carbohydrates (dextrin/DP4/4+) and thereby increase attenuation of the wort.
  • Example 2
  • the pH profile and temperature profile of different pullulanases was analyzed in the present example.
  • the alpha-1 ,6-glycosidic bounds in pullulan were hydrolyzed by a pullulanase enzyme and the increased reducing sugar capacity was detected by a modified Somogyi-Nelson method.
  • Buffer citrate 0.1 M + 0.2 M phosphate (adjusted in the pH profile, pH 5 in temperature profile)
  • Somogyi's cobber reagent Somogyi's cobber reagent. The samples are mixed for 2 minutes followed by addition of water in the same amount as Nelson's reagent. The samples are incubated 30 minutes in the dark and measured in a spectrophotometer at 540 nm.
  • Reagents can be prepared as follows: Somogyi's cobber reagent:
  • pullulanase 3 has a broad pH profile and activity at high temperatures when compared to the other two pullulanases. These properties make pullulanase 3 a very robust enzyme in brewing (mashing conditions), in particular for saccharification temperatures between 62 0 C and 65 0 C.

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Abstract

La présente invention concerne des procédés de production de moût et de bière à partir de malt et d'une mouture complémentaire brassés à une température élevée.
PCT/EP2007/055863 2006-06-15 2007-06-14 Procédé de brassage Ceased WO2007144393A1 (fr)

Priority Applications (1)

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US12/326,326 US8765199B2 (en) 2006-06-15 2008-12-02 Mashing process

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US81394406P 2006-06-15 2006-06-15
US60/813,944 2006-06-15

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PCT/US2007/087209 Continuation-In-Part WO2009075682A1 (fr) 2006-06-15 2007-12-12 Procédé de trempe

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Cited By (12)

* Cited by examiner, † Cited by third party
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BE1020236A3 (nl) * 2011-12-21 2013-06-04 Duvel Moortgat Nv Werkwijze voor het minimaliseren van de thermische belasting in een brouwzaal.
CN103146534A (zh) * 2013-03-28 2013-06-12 济南瑞丰生物工程有限公司 一种固态白酒发酵及生香增强剂的生产方法
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US8765199B2 (en) 2006-06-15 2014-07-01 Novozymes A/S Mashing process
WO2009074650A3 (fr) * 2007-12-12 2009-08-13 Novozymes As Procédé de brassage
US11041139B2 (en) 2007-12-12 2021-06-22 Novozymes A/S Brewing process
CN101952409B (zh) * 2007-12-12 2014-12-10 诺维信公司 酿造方法
WO2009075682A1 (fr) * 2007-12-12 2009-06-18 Novozymes A/S Procédé de trempe
WO2010043538A2 (fr) 2008-10-15 2010-04-22 Novozymes A/S Procédé de brassage
WO2010043538A3 (fr) * 2008-10-15 2011-02-03 Novozymes A/S Procédé de brassage
CN102186965B (zh) * 2008-10-15 2014-10-08 诺维信公司 酿造方法
RU2524118C2 (ru) * 2008-10-15 2014-07-27 Новозимс А/С Способ пивоварения
US11999928B2 (en) 2009-11-13 2024-06-04 Novozymes A/S Brewing method
EP2499227B1 (fr) 2009-11-13 2015-04-08 Novozymes A/S Procédé de brassage
WO2011087836A2 (fr) 2009-12-22 2011-07-21 Novozymes A/S Variants de pullulanase et utilisations de ceux-ci
RU2600885C2 (ru) * 2011-04-15 2016-10-27 Новозимс А/С Способ получения пивного сусла
US10800999B2 (en) 2011-04-15 2020-10-13 Novozymes A/S Method for production of brewers wort
BE1020483A3 (nl) * 2011-12-21 2013-11-05 Duvel Moortgat Nv Werkwijze voor het minimaliseren van de thermische belasting in een brouwzaal.
BE1020236A3 (nl) * 2011-12-21 2013-06-04 Duvel Moortgat Nv Werkwijze voor het minimaliseren van de thermische belasting in een brouwzaal.
CN103146534A (zh) * 2013-03-28 2013-06-12 济南瑞丰生物工程有限公司 一种固态白酒发酵及生香增强剂的生产方法
CN103146534B (zh) * 2013-03-28 2014-03-19 济南瑞丰生物工程有限公司 一种固态白酒发酵及生香增强剂的生产方法
JP2017505129A (ja) * 2014-02-07 2017-02-16 ノボザイムス アクティーゼルスカブ グルコースシロップ生産用組成物
US10337041B2 (en) 2014-02-07 2019-07-02 Novozymes A/S Compositions for producing glucose syrups
EP3712240A1 (fr) * 2014-02-07 2020-09-23 Novozymes A/S Compositions pour la production de sirops de glucose
CN105960176A (zh) * 2014-02-07 2016-09-21 诺维信公司 用于生产葡萄糖糖浆的组合物
WO2015118123A1 (fr) * 2014-02-07 2015-08-13 Novozymes A/S Compositions pour la production de sirops de glucose
US11136607B2 (en) 2014-02-07 2021-10-05 Novozymes A/S Compositions for producing glucose syrups
US11667938B2 (en) 2014-02-07 2023-06-06 Novozymes A/S Compositions for producing glucose syrups
EP4004176B1 (fr) * 2019-07-23 2023-10-18 Heineken Supply Chain B.V. Boisson dérivée d'amidon au goût sucré

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