EP1812547A1 - Procede d'empatage et composition enzymatique utile dans ledit procede - Google Patents

Procede d'empatage et composition enzymatique utile dans ledit procede

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Publication number
EP1812547A1
EP1812547A1 EP05746301A EP05746301A EP1812547A1 EP 1812547 A1 EP1812547 A1 EP 1812547A1 EP 05746301 A EP05746301 A EP 05746301A EP 05746301 A EP05746301 A EP 05746301A EP 1812547 A1 EP1812547 A1 EP 1812547A1
Authority
EP
European Patent Office
Prior art keywords
xylanase
mash
proceeding
wort
mashing
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.)
Withdrawn
Application number
EP05746301A
Other languages
German (de)
English (en)
Inventor
Niels Elvig
Rikke Festersen
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
Original Assignee
Novozymes AS
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 filed Critical Novozymes AS
Publication of EP1812547A1 publication Critical patent/EP1812547A1/fr
Withdrawn legal-status Critical Current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
    • C12C5/00—Other raw materials for the preparation of beer
    • C12C5/004—Enzymes
    • 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
    • C12C7/00—Preparation of wort
    • C12C7/14—Lautering, i.e. clarifying wort
    • C12C7/16—Lautering, i.e. clarifying wort by straining
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01008—Endo-1,4-beta-xylanase (3.2.1.8)

Definitions

  • the present invention relates, inter alia, to a mashing and filtration step in a process for the production of an alcoholic beverage, and to a composition useful in the mashing and filtration step in such a process.
  • the invention provides in a first aspect a process for production of a mash having enhanced filterability and/or improved extract yield after filtration, which comprises; preparing a mash in the presence of enzyme activities and filtering the mash to obtain a wort, wherein the enzyme activities comprise endoglucanase activities derived from Trichoderma Reesei and a xylanase of GH family 10.
  • the invention provides a composition
  • a composition comprising; an endoglucanase derived from Trichoderma reesei; and, a xylanase GH 10 derived from Aspergillus sp., preferably from Aspergillus aculeatus (SEQ ID NO:1).
  • the invention provides uses of a composition according to the second aspect in a process comprising reducing the viscosity of an aqueous solution comprising a starch hydrolysate, in a process comprising filtering of an aqueous solution comprising a starch hydrolysate, in a process wherein the aqueous solution comprising a starch hydrolysate is a mash for beer making and/or in a process wherein the aqueous solution comprising a starch hydrolysate is a feed composition.
  • the final step is to dry the green malt in the kiln.
  • the temperature regime in the kiln determines the colour of the barley malt and the amount of enzymes which survive for use in the mashing process.
  • Low temperature kilning is more appropriate for malts when it is essential to preserve enzymatic activity.
  • Malts kilned at high temperatures have very little or no enzyme activity but are very high in colouring such as caramelized sugars as well as in flavouring compounds.
  • 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 malt 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.
  • step- infusion This mashing procedure involves a series of rests at various temperatures, each favouring one of the necessary endogenous enzyme activities.
  • step- infusion To day the double-mash infusion system is the most widely used system for industrial production of beer, especially lager type beer. This system prepares two separate mashes. It utilizes a cereal cooker for boiling adjuncts and a mash tun for well-modified, highly enzymatically active malts.
  • mashing may be performed in the presence of added enzyme compositions comprising the enzymes necessary for the hydrolysis of the grist starch.
  • These enzymes may comprise alpha- amylases, pullulanases, beta-amylases and glucoamylases.
  • the liquid extract the wort
  • the solids spent grains i.e. the insoluble grain and husk material forming part of grist.
  • Wort separation is important because the solids contain large amounts of non-starch polysaccharides, protein, poorly modified starch, fatty material, silicates, and polyphenols (tannins).
  • Important non-starch polysaccharides present in cereal grains are beta-glucan and arabinoxylan.
  • the endosperm cell wall of barley comprises 75% beta-glucan, 20% arabinoxylan, and 5% remaining protein with small amount of cellulose, glucomannan and phenolic acids.
  • Long chains of barley arabinoxylans, and to a lesser degree beta-glucan, which have not been modified due to enzymatic hydrolysis may cause formation of gels when solubilised in water, these gels will strongly increase wort viscosity and reduce filterability.
  • beta-glucan has been reduced to smaller oligomers, as unmodified beta-glucans later on will give rise to haze stability problems in the final beer.
  • enzymatic compositions comprising endoglucanases and xylanases, such as Ultraflo® or Viscozyme®, are often used in the mashing step to improve wort separation.
  • the objectives of wort separation include the following: • to obtain good extract recovery, • to obtain good filterability, and • to produce clear wort. Extraction recovery and filterability are important for the economy in the brewing process, while the wort clarity is a must in order to produce a beer which does not develop haze. Extraction recovery, filterability and wort clarity is greatly affected by the standard of the grist, e.g. the barley malt and the types of adjunct, as well as the applied mashing procedure.
  • the wort may be fermented with brewers yeast to produce a beer.
  • 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.
  • 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 be any carbohydrate rich material.
  • mash is understood as a aqueous starch slurry, e.g. comprising crushed barley malt, crushed barley, and/or other adjunct or a combination hereof, steeped in water to make wort.
  • wort is understood as the unfermented liquor run-off following extracting the grist during mashing.
  • spent grains is understood as the drained solids remaining when the grist has been extracted and the wort separated from the mash.
  • beer is here understood as fermented wort, e.g. an alcoholic beverage brewed from barley malt, optionally adjunct and hops.
  • extract recovery in the wort is defined as the sum of soluble substances extracted from the grist (malt and adjuncts) expressed in percentage based on dry matter.
  • a thermostable enzyme is understood as an enzyme that under the temperature regime and the incubation period applied in the processes of the present invention in the amounts added is capable of sufficient degradation of the substrate in question.
  • homology when used about polypeptide or DNA sequences and referred to in this disclosure is understood as the degree of homology between two sequences indicating a derivation of the first sequence from the second.
  • the homology 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 53711 ) (Needleman, S.B. and Wunsch, CD., (1970), Journal of Molecular Biology, 48, 443-453.
  • GAP GAP creation penalty of 3.0
  • GAP extension penalty 0.1.
  • DP is the degree of polymerisation, herein used for average number of glucose units in polymers in a polysaccharide hydrolysate.
  • GH Glycoside Hydrolase Families
  • Binding Modules applied in this disclosure follows the concept of Coutinho, P.M. & Henrissat, B. (1999) CAZy - Carbohydrate-Active Enzymes server at URL: http://afmb.cnrs- mrs.fr/ ⁇ cazy/CAZY/index.html or alternatively Coutinho, P.M. & Henrissat, B. 1999;
  • Genetics, Biochemistry and Ecology of Cellulose Degradation K. Ohmiya, K. Hayashi, K. Sakka, Y. Kobayashi, S. Karita and T.
  • composition according to the proceeding aspect may be used in a process comprising reducing the viscosity of an aqueous solution comprising a starch hydrolysate.
  • the composition may even be used in a process comprising filtering of an aqueous solution comprising a starch hydrolysate.
  • the aqueous solution comprising a starch hydrolysate is a mash for beer making, and in another preferred embodiment the aqueous solution comprising a starch hydrolysate is a feed composition.
  • the process of the invention may be applied in the mashing of any grist.
  • the grist may comprise any starch and/or sugar containing plant material derivable from any plant and plant part, including tubers, roots, stems, leaves and seeds.
  • the grist comprises grain, such as grain from barley, wheat, rye, oat, corn, rice, milo, millet and sorghum, and more preferably, at least 10%, or more preferably at least 15%, even more preferably at least 25%, or most preferably at least 35%, such as at least 50%, at least 75%, at least 90% or even 100% (w/w) of the grist of the wort is derived from grain.
  • the grist comprises malted grain, such as barley malt.
  • At least 10%, or more preferably at least 15%, even more preferably at least 25%, or most preferably at least 35%, such as at least 50%, at least 75%, at least 90% or even 100% (w/w) of the grist of the wort is derived from malted grain.
  • the mashing enzymes may be exogenously supplied.
  • the enzymes mostly used as starch degrading enzymes include pullulanases, alpha- amylases and amyloglucosidases. The use of starch degrading enzymes in mashing is well- known to the skilled person.
  • Adjunct comprising readily fermentable carbohydrates such as sugars or syrups may be added to the malt mash before, during or after the mashing process of the invention but is preferably added after the mashing process.
  • a part of the adjunct may be treated with a protease and/or an endoglucanase, and/or heat treated before being added to the mash of the invention.
  • starch extracted from the grist is gradually hydrolyzed into fermentable sugars and smaller dextrins.
  • the mash is starch negative to iodine testing, before wort separation.
  • the application of the appropriate xylanase and endoglucanase activities in the process of the present invention results in efficient reduction of beta-glucan and arabino- xylan level facilitating wort separation, thus ensuring reduced cycle time, high extract recovery and clear wort.
  • the wort produced by the process of the first aspect of the invention may be fermented to produce a 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.
  • the processes 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.
  • the beer produced by fermenting the wort of the invention may be any type of beer, e.g. ale, strong ale, stout, porter, lager, pilsner, bitter, export beer, malt liquor, happoushu, Iambic, barley wine, high- alcohol beer, low-alcohol beer, low-calorie beer or light beer.
  • the beer produced by the process of the invention may be distilled to recover ethanol, e.g. for whisky production.
  • Contemplated are any kind of whisky (spelled "whiskey” in US and Ireland) include bourbon, Canadian whisky, Irish whiskey, rye, and scotch.
  • xylanase For the present purposes a xylanase is an enzyme classified as EC 3.2.1.8. The official name is endo-1 ,4-beta-xylanase. The systematic name is 1 ,4-beta-D-xylan xylanohydrolase.
  • endo-(1-4)-beta-xylanase (1-4)-beta- xylan 4-xylanohydrolase; endo-1, 4-xylanase; xylanase; beta-1 ,4-xylanase; endo-1 ,4- xylanase; endo-beta-1 ,4-xylanase; endo-1, 4-beta-D-xylanase; 1 ,4-beta-xylan xylanohydrolase; beta-xylanase; beta- 1 ,4-xy Ian xylanohydrolase; endo-1 ,4-beta-xylanase; beta-D-xylanase.
  • the reaction catalysed is the endohydrolysis of 1 ,4-beta-D-xylosidic linkages in xylans.
  • the xylanase to be used for the present invention may be of any origin including mammalian, plant or animal origin it is presently preferred that the xylanase is of microbial origin.
  • the xylanase may be one derivable from a filamentous fungus or a yeast.
  • Xylanases have been found in a number of fungal species, in particular species of Aspergillus, such as A. niger, A. awamori, A. aculeatus and A. oryzae, Trichoderma, such as T.
  • xylanases have also been found in bacterial species, e.g. within the genus Bacillus, such as B. pumilus.
  • the xylanase is derived from a filamentous fungus such as from Aspergillus sp., Bacillus sp., Humicola sp., Myceliophotora sp., Poitrasia sp.
  • the xylanase to be used in the present invention is a Glycoside Hydrolase Family 10 (GH10) xylanase.
  • GH10 Glycoside Hydrolase Family 10
  • the xylanase to be used according to the process of the invention the xylanase from Aspergillus aculeatus shown as SEQ ID NO:1 (XYL II) in the present disclosure and/or described in WO9421785A1 and comprising the partial amino acid sequence shown as SEQ ID NO:5 therein.
  • any xylanase having and or comprising a sequence having at least 50%, at least 60%, at least 70%, at least 80%, or even at least 90% homology to any of the aforementioned sequences.
  • Endoglucanases For the present purposes an endoglucanase is an enzyme classified as EC 3.2.1.4.
  • the endoglucanase activity to be used for the present invention may be any composition derived from Trichoderma reesei, preferably the composition is obtained by submerged fermentation, such as the composition Celluclast® available from Novozymes A S. Celluclast has a pronounced viscosity-reducing effect on soluble cellulosic substrates.
  • the cellulase composition Laminex BG® a commercial cellulase preparation produced by Trichoderma reesei and available from Genencor International Inc.
  • the xylanolytic activity can be expressed in FXU(S)-units, determined at pH 6.0 with
  • Azo-Wheat arabinoxylan (arabinoxylan dyed with remazol brilliant blue, Megazyme) as substrate.
  • a xylanase sample is incubated with the remazol-xylan substrate.
  • the background of non-degraded dyed substrate is precipitated by ethanol.
  • the remaining blue colour in the supernatant (as determined spectrophotometrically at 585 nm) is proportional to the xylanase activity, and the xylanase units are then determined relatively to an enzyme standard at standard reaction conditions, i.e.
  • Endoclucanase activity The cellulytic activity may be measured in fungal endoglucanase units (FBG), determined on a 0.5% beta-glucan substrate at 30°C, pH 5.0 and reaction time 30 min.
  • Fungal endoglucanase reacts with beta-glucan releases glucose or reducing carbohydrate which is determined as reducing sugar according to the Somogyi-Nelson method.
  • 1 fungal endoglucanase unit (FBG) is the amount of enzyme which according to the above outlined standard conditions, releases glucose or reducing carbohydrate with a reduction capacity equivalent to 1 micromol glucose per minute.
  • the cellulytic activity may be measured in endo-glucanase units (EGU), determined at pH 6.0 with carboxymethyl cellulose (CMC) as substrate.
  • EGU endo-glucanase units
  • a substrate solution is prepared, containing 34.0 g/l CMC (Hercules 7 LFD) in 0.1 M phosphate buffer at pH 6.0.
  • the enzyme sample to be analyzed is dissolved in the same buffer. 5 ml substrate solution and 0.15 ml enzyme solution are mixed and transferred to a vibration viscosimeter (e.g. MIVI 3000 from Sofraser, France), thermostated at 40DC for 30 minutes.
  • One EGU is defined as the amount of enzyme that reduces the viscosity to one half under these conditions.
  • the amount of enzyme sample should be adjusted to provide 0.01-0.02 EGU/ml in the reaction mixture.
  • the arch standard is defined as 880 EGU/g.
  • Enzymes A Celluclast ® 1.5 L, a commercial preparation produced by Trichoderma reesei comprising cellulase activities 700 EGU/g and xylanase activities 200 FXU(S)/g.
  • B composition of the invention comprising the cellulase and xylanase activities, respectively 700 EGU/g and 200 FXU(S)/g, from Trichoderma reesei and the GH10 xylanase from Aspergillus aculeatus shown in SEQ ID NO:1 250 FXU(S)/g.
  • C Laminex BG®, a commercial preparation produced by Trichoderma reesei comprising cellulase activities 1370 EGU/g and xylanase activities.
  • the mash was prepared according to EBC: 4.5.1 using malt grounded according to EBC: 1.1. Mashing trials were performed in 500 ml lidded vessels incubated in water bath with stirring and each containing a mash with 44 g malt and adjusted to a total weight of 300 ⁇ 0.2 g with water preheated to 50°C. The wort produced was app. 12% Plato.
  • Mashing temperature profile Unless otherwise stated mashing was carried out using an initial incubation temperature at 50°C for 30 minutes, followed by an 17 min increasing step with 1°C/min to 67°C remaining here for 40 min. The profile is continued with an 8 min increasing step with
  • E, - 100 b)E 2 100 - M
  • E-, the extract content of sample
  • E 2 the extract content of dry grist
  • P the extract content in wort
  • Plato M the moisture content of the grist
  • in % (m/m) 800 the amount of destilled water added into the mash to 100 g of grist
  • Viscosity Automated Microviscometer (AMVn) is based on the rolling ball principle.
  • the sample to be measured is introduced into a glass capillary in which a steel ball rolls.
  • the viscous properties of the test fluid can be determined by measuring the rolling time of the steel ball.
  • the rolling time t 0 of a ball over a defined measuring distance in a capillary is measured.
  • the dynamic viscosity ⁇ of the sample is calculated from the calibration constant K ⁇ ( ⁇ ) of the measuring system, the rolling time to and the difference of density ⁇ p between the ball and the sample.
  • K ⁇ ( ⁇ ) of the measuring system K ⁇ ( ⁇ ) of the measuring system
  • ⁇ 1 K ⁇ ⁇ a) • f Q • (p k - p s ),
  • ⁇ Dynamic vis cos ity of the sample
  • Example 1 A mash was prepared from a well modified malt (172 mg ⁇ -glucan/l). Four treatments according to table 1 were applied. The results are presented in table 2 The treatment B wherein the enzyme composition comprised a GH10 xylanase had a significantly better performance on viscosity reduction than blank, as well as than the treatments A and C consisting only of a T. reesei preparation. The differences are significant an at least the 99.9% level.

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Food Science & Technology (AREA)
  • Enzymes And Modification Thereof (AREA)
  • Distillation Of Fermentation Liquor, Processing Of Alcohols, Vinegar And Beer (AREA)

Abstract

La présente invention concerne une étape d'empâtage et de filtration dans un procédé de brassage ainsi qu'une composition comprenant une endoglucanase dérivée à partir de Trichoderma reesei et une xylanase GH10 dérivée à partir d'Aspergillus, utile dans l'étape d'empâtage et de filtration d'un procédé de brassage.
EP05746301A 2004-06-03 2005-06-02 Procede d'empatage et composition enzymatique utile dans ledit procede Withdrawn EP1812547A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DKPA200400870 2004-06-03
PCT/DK2005/000367 WO2005118769A1 (fr) 2004-06-03 2005-06-02 Procede d'empatage et composition enzymatique utile dans ledit procede

Publications (1)

Publication Number Publication Date
EP1812547A1 true EP1812547A1 (fr) 2007-08-01

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WO2010118257A2 (fr) * 2009-04-08 2010-10-14 Danisco Us Inc. Endoglucanase destinée à réduire la viscosité d'une boue de substances végétales
US9012186B2 (en) 2009-04-27 2015-04-21 The Board Of Trustees Of The University Of Illinois Hemicellulose-degrading enzymes
PL2427550T3 (pl) * 2009-05-07 2019-09-30 Dupont Nutrition Bioscences Aps Kompleks enzymatyczny z enzymów trichoderma reesei i p. funiculosum
WO2011038019A2 (fr) 2009-09-23 2011-03-31 Danisco Us Inc. Nouvelles enzymes glycosyl hydrolases et utilisations de celles-ci
CN102639697B (zh) 2009-11-06 2015-03-25 诺维信股份有限公司 具有木聚糖酶活性的多肽和编码该多肽的多核苷酸
CN102686736B (zh) 2009-12-23 2017-05-31 丹尼斯科美国公司 提高同步糖化发酵反应效率的方法
JP6290626B2 (ja) 2011-03-17 2018-03-14 ダニスコ・ユーエス・インク 糖化プロセスにおける粘度を低下させる方法
PL2756078T3 (pl) 2011-09-14 2017-12-29 Dupont Nutrition Biosciences Aps Kompozycje zawierające enzymy o aktywności endo-1,4-beta-ksylanazy i enzymy o aktywności endo-1, 3(4)-beta glukanazy
AU2012387042A1 (en) 2012-08-03 2015-02-19 Dupont Nutrition Biosciences Aps Enzymes
RU2695461C2 (ru) * 2013-09-05 2019-07-23 Новозимс А/С Способ снижения вязкости в способе пивоварения
EP3017706A1 (fr) 2014-11-05 2016-05-11 Dupont Nutrition Biosciences ApS Enzymes de maltage

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CA2586779A1 (fr) 2005-12-15
WO2005118769A1 (fr) 2005-12-15

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