WO2024078714A1 - Procédé de production de moût de brasserie - Google Patents

Procédé de production de moût de brasserie Download PDF

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Publication number
WO2024078714A1
WO2024078714A1 PCT/EP2022/078390 EP2022078390W WO2024078714A1 WO 2024078714 A1 WO2024078714 A1 WO 2024078714A1 EP 2022078390 W EP2022078390 W EP 2022078390W WO 2024078714 A1 WO2024078714 A1 WO 2024078714A1
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Prior art keywords
sorghum
alpha
unmalted
grist
amylase
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PCT/EP2022/078390
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English (en)
Inventor
Alexander Mauch
Carsten Andersen
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Novozymes A/S
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Priority to PCT/EP2022/078390 priority Critical patent/WO2024078714A1/fr
Publication of WO2024078714A1 publication Critical patent/WO2024078714A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2414—Alpha-amylase (3.2.1.1.)
    • C12N9/2417—Alpha-amylase (3.2.1.1.) from microbiological source
    • 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
    • C12C7/047—Preparation or treatment of the mash part of the mash being unmalted cereal mash
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2405—Glucanases
    • C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
    • C12N9/2411—Amylases
    • C12N9/2414—Alpha-amylase (3.2.1.1.)
    • C12N9/2417—Alpha-amylase (3.2.1.1.) from microbiological source
    • C12N9/242—Fungal source
    • 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)
    • C12Y302/01001—Alpha-amylase (3.2.1.1)

Definitions

  • the present invention relates to a method for production of a brewer’s wort using sorghum in the grist.
  • FIG 1 Figure 1 shows an overview of the liquefaction process as run on the Rapid Visco Analyzer RVA 4500 (see Materials and Methods, Liquefaction assay).
  • the top line is the Temperature curve, and indicates the temperature over time.
  • the bottom line indicates the viscosity measurements taken over time.
  • the performance indicators derived from in-situ viscosity measurements taken over the course of the liquefaction experiment are highlighted.
  • the shaded area VA indicates the Viscosity Area (Area under Viscosity curve).
  • the time span used for calculating the area is indicated by arrows.
  • the measurement at point VBC is the viscosity before cooling.
  • the final viscosity is indicated by FV and is measured at the very end of the process, after cooling.
  • Set-back (SB) is measured as VBC (viscosity before cooling) - FV (final viscosity), (see Materials and Methods, Tables 3A; as well as Example 1 and Table 4).
  • FIG 2 Figure 2 shows Final viscosities after the liquefaction process of sorghum in presence of different alpha-amylases Y-axis Viscosity measured in cP (centipoise); x-axis for the different enzymes tested. (See Example 1 and Table 4).
  • FIG 3 Figure 3 shows Area underneath viscosity curve from 10 min until end, derived from integration of the individual viscosity measurements during the liquefaction process of sorghum in presence of different alpha-amylases tested in Example 2 (see Example 1 and Table 4).
  • FIG 4 Figure 4 shows the Set-back values for the samples from Example 1 (see Example 1 and Table 4).
  • X-axis Enzymes tested;
  • y-axis viscosity measured in cP.
  • enzymes are often added as a supplement when mashing malt and adjunct grist. Enzymes may also be applied in mashing of well modified malts with high enzyme content to increase the extract recovery and the amount of fermentable sugars, as well as accelerate the overall conversion time.
  • WO 2012/140075 describes the use of a glucoamylase from Penicillium oxalicum when mashing a malt and adjunct grist.
  • alpha-amylases with calculated pl in the range from 4.0 to 4.6 are particularly efficient in liquifying sorghum, even in sorghum with high content of condensed tannins (sorghum comprising at least 10mg catechin equivalents/g sorghum).
  • the invention relates in a first aspect to a method of producing a brewer’s wort comprising the steps a) Providing a mash from a grist comprising unmalted sorghum; b) Adding an alpha-amylase to the mash; c) Heating the mash to the pasting temperature of the unmalted sorghum starch, and d) Producing a wort, wherein the alpha amylase has a calculated pl in the range of from 4.0 to 4.6.
  • the invention relates in a second aspect to the use of an alpha-amylase with a calculated pl in the range of from 4.0 to 4.6, for brewing, in particular sorghum brewing.
  • the invention relates to a beer produced according to the method.
  • grist is understood as the starch- or sugar-containing material that is the basis for beer production.
  • malt is understood as any malted cereal grain.
  • amalgamate is understood as the part of the grist which is not malt.
  • mash is understood as a starch containing slurry of the grist comprising crushed grain, optionally other starch containing material, or a combination thereof, steeped in water to make wort.
  • mashing is the process of converting starch in the mash into fermentable and un-fermentable sugars.
  • wort is understood as the unfermented liquor run-off, following extracting the grist during mashing.
  • identity is the relatedness between two amino acid sequences.
  • degree of identity is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends in Genetics 16: 276-277), preferably version 5.0.0 or later.
  • the optional parameters used are gap open penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix.
  • the output of Needle labeled “longest identity” (obtained using the -nobrief option) is used as the percent identity and is calculated as follows:
  • the invention relates in a first aspect to a method of producing a brewer’s wort comprising the steps: a) Providing a mash from a grist comprising unmalted sorghum; b) Adding an alpha-amylase to the mash; c) Heating the mash to the pasting temperature of the unmalted sorghum starch; and d) Producing a wort wherein the alpha-amylase has a calculated pl in the range from 4.0 to 4.6.
  • the method of the invention improves yield where unmalted sorghum is used in brewing as it leads to better usage of the raw material.
  • the method of the invention further makes it possible to use sorghum cultivars not previously possible to be used in brewing (sorghum containing high content of condensed tannins, e.g. “bird-proof” cultivars of sorghum). Even further, the method of the invention provides an advantage as brewers may use the same method regardless of tannin content in sorghum, obviating the need for switching or running alternative methods.
  • the grist is mixed with water prior to mashing.
  • the water may, before being added to the grist, be preheated in order for the mash to attain the desired mash temperature at the moment of mash forming.
  • additional heat is preferably supplied in order to attain the desired start process temperature.
  • the desired start mashing temperature is attained within 15 minutes, or more preferably within 10 minutes, such as within 9, 8, 7, 6, 5, 4, 3, 2 minutes or even more preferably within 1 minute after the mash forming, or most preferably the desired mashing temperature is attained at the mash forming.
  • the alpha amylase may be added to the mash ingredients, e.g., the water and/or the grist before, during or after forming the mash, or at any time during the mashing.
  • the alpha amylase may be added as an enzyme composition.
  • Such enzyme compositions may comprise one or more enzymes.
  • the enzyme composition in addition to the enzyme(s), may also further comprise at least one other substance, for example but not limited to buffer, surfactants etc.
  • the enzyme compositions may be in any form, for example, solid, liquid, emulsion, gel, or paste. Such forms are known to the person skilled in the art.
  • starch extracted from the grist is gradually hydrolysed into fermentable sugars, smaller dextrins, and glucose.
  • the mashing comprises as a step of heating, suitable to achieve pasting and/or gelatinization.
  • the method of the invention comprises a step of heating the mash to the pasting temperature of the unmalted sorghum starch. This is done in order to achieve at least pasting and/or gelatinization of the sorghum starch. RVA determined pasting temperature can be used as an indication of starch gelatinization.
  • the pasting/gelatinization temperature of sorghum is known in the art, and is typically in the range from 65 to 78°C.
  • this heating step may be at a temperature of at least 60°C, at least 65°C, or at least 70°C, at least 75°C, at least 78°C, at least 79°C or at least 80°C; or for example at a temperature in the range of from 60 to 95°C, such as from 62 to 95°C, 67 to 95°C, 70 to 95°C, 72 to 95°C, 75 to 95°C, 77 to 95°C, 80 to 95°C, 82 to 95°C or 85 to 95°C, for a time which is suitable to achieve pasting.
  • the heating step may for example be at any temperature interval mentioned above and for a time of at least 5 minutes, such as at least 7 min, at least 9 min, at least 10 min, at least 12 min, at least 15 min, at least 20 min; or for example for a time in the range of from 2 to 90 min, such as 2 to 85 min, 2 to 80 min, 2 to 75 min, 2 to 70 min, 2 to 65 min, 2 to 60 min.
  • Particular embodiments relate to an incubation at at least 60°C for at least 5 mins, or for example at at least 65°C for at least 10 mins, or at a temp in the range of from 60 to 95°C for a time in the range of from 5 to 90 mins; or a temperature in the range of from 65 to 90°C for a time in the range of from 5 to 80 mins.
  • heating step is at a temperature in the range of from 70 to 95°C for a time in the range of from 5 to 40 mins.
  • Wort separation, lautering is important because the solids contain large amounts of protein, poorly modified starch, fatty material, silicates, and polyphenols (tannins).
  • the extract retained in the spent grain after collection of the first wort may also be washed out by adding hot water on top of the lauter cake. This process is called sparging.
  • the hot water flows through the spent grain and dissolves the remaining extract.
  • the diluted wort is called second wort.
  • the wort is boiled, optionally after addition of hops.
  • the wort After cooling and removal of the precipitates, the wort is aerated, and yeast is added.
  • the yeast applied may be any yeast suitable for beer brewing, especially yeasts selected from Saccharomyces such as S. cerevisiae and S. uvarum, including natural or artificially produced variants of these organisms.
  • yeasts selected from Saccharomyces such as S. cerevisiae and S. uvarum, including natural or artificially produced variants of these organisms.
  • a person skilled in the art will be able to select a suitable yeast.
  • Any beer type may be produced from the wort according to the present invention.
  • Preferred beer types comprise 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 wort may also be processed to be used as syrups. It may also be used to produce non- alcoholic beverages. These processes are well known to a person skilled in the art.
  • the method according to the present invention comprises providing a mash from a grist comprising unmalted sorghum.
  • Sorghum belongs to the family of grasses (Poaceae) and comes in different cultivars and varieties that are adapted for various areas and uses. Insight into the specific physicochemical properties and how physical- and biochemical treatments affect them is the basis to assure an efficient processability in a brewery and hence good beer quality.
  • the method of brewing according to the invention provides a robust method for brewing sorghum, regardless of condensed tannin content.
  • the unmalted sorghum may be one or more species selected from the following:
  • Sorghum leiocladum 18. Sorghum macrospermum
  • the sorghum is Sorghum bicolour
  • the sorghum is Sorghum bicolor (L.) Moench.
  • the invention relates in some embodiments to where the grist comprises at least 1% unmalted sorghum, such as at least 2% unmalted sorghum, such as at least 4% (w/w) unmalted sorghum, such as at least 5% (w/w) unmalted sorghum; or for example the grist comprises in the range of from 1 to 100% unmalted sorghum, such as from 1 to 90%, 2 to 90%, 2 to 85%, 2 to 80%, 2 to 85%% unmalted sorghum (w/w).
  • the method according to the invention provides improved liquefaction of sorghum.
  • the grist comprises at least 10% w/w unmalted sorghum, such as at least 15% w/w unmalted sorghum, at least 20% w/w unmalted sorghum, as at least 25% w/w unmalted sorghum, at least 30% w/w unmalted sorghum, at least 35% w/w unmalted sorghum; such as the grist comprises at least 40% (w/w) unmalted sorghum, e.g. the grist comprises at least 45% (w/w) unmalted sorghum; e.g., the grist comprises at least 50% (w/w) unmalted sorghum; e.g.
  • the grist comprises at least 55% (w/w) unmalted sorghum; e.g., the grist comprises at least 60% (w/w) unmalted sorghum; e.g., the grist comprises at least 70% (w/w) unmalted sorghum; e.g. the grist comprises at least 75% (w/w) unmalted sorghum; e.g.
  • the grist comprises at least 80% (w/w) unmalted sorghum; e.g., the grist comprises at least 85%(w/w) unmalted sorghum; e.g., the grist comprises at least 90% (w/w) unmalted sorghum; e.g., the grist comprises at least 95% (w/w) unmalted sorghum.
  • the grist consists of unmalted sorghum.
  • the grist comprises both unmalted sorghum and malted sorghum.
  • the grist comprising unmalted sorghum may further comprise one or more malted or unmalted cereal and/or pseudocereal grains, such as barley, wheat, rye, buckwheat, amaranth, quinoa, millet, oat, sorghum or combinations thereof.
  • the grist comprises or consists of unmalted sorghum and malted barley.
  • the grist may comprise further adjunct, such as raw and/or refined starch and/or sugar containing material derived from one or more of wheat, rye, oat, maize, rice, milo, millet, sorghum, potato, sweet potato, cassava, tapioca, sago, banana, sugar beet and/or sugar cane.
  • raw and/or refined starch and/or sugar containing material derived from one or more of wheat, rye, oat, maize, rice, milo, millet, sorghum, potato, sweet potato, cassava, tapioca, sago, banana, sugar beet and/or sugar cane.
  • the grist comprises or consists of unmalted sorghum and cassava.
  • said further adjunct(s) have high pasting or gelatinization temperature, such as a temperature of at least 60°C. More particularly, these adjuncts have a high onset pasting or gelatinization temperature.
  • Adjunct may also comprise readily fermentable carbohydrates such as sugars or syrups and they may be added to the mash before, during or after the mashing process of the invention, but is preferably added after the mashing process.
  • the adjunct Prior to forming the mash, the adjunct is preferably milled and most preferably dry or wet milled.
  • Condensed tannins proanthocyanidins, polyflavonoid tannins, catechol-type tannins, pyrocatecollic type tannins, non-hydrolyzable tannins or flavolans
  • Condensed tannins are polymers formed by the condensation of flavans.
  • the total amount unmalted sorghum in the grist may have a content of condensed tannins, as determined by vanillin method (see materials and methods) in the range of from 0.2 mg to 90 mg catechin equivalents per g sorghum, for example in the range of from 1 to 90, from 3 to 90, from 5 to 90, from 6 to 90, from 10 to 90, from 15 to 90, from 18 to 90, from 20 to 90, from 25 to 90, from 30 to 90, from 35 to 90, from 40 to 90, from 45 to 90, from 50 to 90, from 50 to 90, from 60 to 90, from 65 to 90, from 70 to 90 mg catechin equivalents per g sorghum, as determined by vanillin assay (see Materials and Methods).
  • Sorghum are sometimes divided into three groups based on tannin content (Hahn and Rooney, Cereal Chem., 63(1).4-8) 1985).
  • Group I sorghums do not contain tannins; while Group II and Group III do.
  • Group III contains more tannins than do Group II sorghums.
  • Group III sorghum is sometimes known as “birdproof cultivars”. So-called birdproof cultivars and varieties of sorghum show superior agronomic characteristics and are therefore the preferred choice of farmers in regions, where the grain is exposed to migration birds. The high content of condensed tannins in these varieties renders the sorghum less attractive to birds.
  • the method according to the invention is useful for liquefaction of all sorghum, and in particular, surprisingly, is efficient on sorghum having high content of condensed tannins.
  • some embodiments relate to the method according to the invention, wherein the unmalted sorghum comprises or consists of sorghum having high content of condensed tannins.
  • high content of condensed tannins refers to sorghum having at least 10 mg catechin equivalents/ g sorghum.
  • the unmalted sorghum may comprise or consist of one or more Group III sorghum.
  • the unmalted sorghum may comprise or consist of one or more Group II sorghum.
  • the unmalted sorghum does not comprise sorghum from Group I.
  • the unmalted sorghum provided consists of sorghum from Group II and/or Group III.
  • the unmalted sorghum provided consists of sorghum having at least 10 mg catechin equivalents/ g sorghum.
  • Alpha-amylase (EC 3.2.1.1)
  • alpha-amylases with a calculated pl in the range from 4.0 to 4.6 are particularly efficient at liquefying sorghum, in particular sorghum having high content of condensed tannins.
  • the invention relates to a method of producing a brewer’s wort comprising the steps a) Providing a mash from a grist comprising unmalted sorghum; b) Adding an alpha-amylase to the mash; c) Heating the mash to the pasting temperature of the unmalted sorghum, and d) Producing a wort, wherein the alpha amylase has a calculated pl in the range of from 4.0 to 4.6.
  • Some embodiments relate to the method according to the invention wherein the alphaamylase according the embodiment has a calculated pl in the range of from 4.0 to 4.6, such as 4.1 to 4.6, or for example from 4.2 to 4.5.
  • the alpha amylase has at least 60% identity, such as at least 65% identity, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% or even 100% identity to the sequence shown in SEQ ID NO: 1.
  • the alpha amylase has at least 60% identity, such as at least 65% identity, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% or even 100% identity to the sequence shown in SEQ ID NO: 5
  • the alpha amylase has at least 60% identity, such as at least 65% identity, such as at least 75%, such as at least 80%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% or even 100% identity to the sequence shown in SEQ ID NO:9.
  • the alpha amylase according to the invention is suitable for use at or above pasting temperature for sorghum.
  • the alpha amylase according to the invention displays at least 50% residual activity at pasting temperature after 10 minutes.
  • the alpha amylase according to the invention such as displays at least 50% residual activity after 10 minutes at a temperature of 60°C, 65°C, 70°C, 75°C, 78°C, 79°C , 80°C, 85 °C, 90°C or 95°C ; or for example at a temperature in the range of from 60 to 95°C, such as from 62 to 95°C, 67 to 95°C, 70 to 95°C, 72 to 95°C, 75 to 95°C, 77 to 95°C, 80 to 95°C, 82 to 95°C or 85 to 95°C.
  • the alpha amylase according to the invention displays at least 50% residual activity after 10 minutes at a temperature of at least 78°C, such as at a temperature of at least 80°C, at least 85°C or at least 90°C.
  • the brewer may choose to employ one or more further alpha-amylases.
  • some embodiments relate to the method according to the invention, wherein one or more further alpha-amylases are added.
  • the one or more further alpha-amylases are not limited, and the selection of such amylases is within the skill of the art.
  • Such one or more further alpha amylases may be added prior to, at the same time as, or after the addition of the alpha-amylase according to the invention.
  • Some embodiments relate to where the one or more further alpha amylases are selected from the group consisting of Amylex 6T (Dupont), Termamyl SC (Novozymes), Fortiva Revo (Novozymes).
  • alpha amylase comprises ENZ 1 and said further alpha-amylases is selected from one or more of ENZ 2, ENZ 3, ENZ 4, ENZ 6, ENZ 7, ENZ 8, ENZ 10, ENZ 11 , ENZ 12, molecules having at least 70% sequence identity to these, and combinations thereof.
  • adding alpha amylase comprises adding ENZ 1, ENZ 5 and/or ENZ 9, or a molecule having at least 60% sequence identity to them, or combinations thereof; and further comprises adding one or more further alpha-amylases, such as one or more of commercially available alphaamylases, such as Amylex 6T (Dupont), Fortiva Revo (Novozymes), Termamyl (Novozymes).
  • adding alpha amylase comprises adding ENZ 1, ENZ 5 and/or ENZ 9, or a molecule having at least 60% sequence identity to them, or combinations thereof; and further comprises adding one or more further alpha-amylases, such as one or more of commercially available alphaamylases, such as Amylex 6T (Dupont), Fortiva Revo (Novozymes), Termamyl (Novozymes).
  • adding alpha amylase comprises adding ENZ 1 or a molecule having at least 60% sequence identity to SEQ ID NO: 1, and further comprises adding one or more further alpha-amylases, such as one or more of commercially available alpha-amylases, such as Amylex 6T (Dupont), Fortiva Revo( Novozymes), Termamyl SC(Novozymes).
  • adding alpha amylase comprises adding ENZ 1 or a molecule having at least 60% sequence identity to SEQ ID NO: 1, and further comprises adding one or more further alpha-amylases, such as one or more of commercially available alpha-amylases, such as Amylex 6T (Dupont), Fortiva Revo( Novozymes), Termamyl SC(Novozymes).
  • alpha amylase is added in a concentration of 0.5 to 500 mg of enzyme protein (EP) per kg of total weight of the grist, e.g., 5 to 500 mg of enzyme protein (EP) per kg of total weight of the grist, e.g., 10 to 100 mg of enzyme protein (EP) per kg of total weight of the grist.
  • EP enzyme protein
  • Glucoamylases are enzymes which catalyze the hydrolysis of terminal (1-4)-linked alpha-D-glucose residues successively from non-reducing ends of the chains with release of beta-D-glucose.
  • the method of the invention further comprises addition of one or more glucoamylases.
  • said one or more glucoamylases may be for example as described in W02019219601, hereby incorporated by reference.
  • one or more further enzyme(s) is added to the mash, said enzyme(s) including but not limited to isoamylase, protease, cellulase, glucanase, laccase, xylanase, lipase, phospholipolase, phytase, phytin, and esterase.
  • the further enzyme added includes but is not limited to a protease.
  • the further enzyme added includes but is not limited to a cellulase.
  • the further enzyme added includes but is not limited to a xylanase.
  • the further enzyme added includes but is not limited to a lipase.
  • the invention relates to the use of an alpha-amylase with a calculated pl in the range from 4.0 to 4.6, for use in brewing, in particular in brewing of beer comprising sorghum.
  • alpha amylase for use in brewing of beer comprising sorghum, wherein said alpha amylase is selected from the group consisting of ENZ1, ENZ 5 and ENZ 9 and sequences with homology of at least 60% thereto.
  • the method of the invention liquefies the sorghum mash and displays little to no setback. Indeed in experimental conditions, negative set-back is shown. This facilitates the handling of the mash.
  • a further aspect of the invention relates to a mash produced by the method according to the invention as described herein, comprising an alpha amylase according to the invention.
  • the invention in a further aspect relates to a beer produced according to the method of the invention.
  • a method of producing a brewer’s wort comprising the steps a) Providing a mash from a grist comprising unmalted sorghum; b) Adding an alpha-amylase to the mash; c) Heating the mash to the pasting temperature of the unmalted sorghum starch, and d) Producing a wort, wherein the alpha amylase has a calculated pl in the range of from 4.0 to 4.6.
  • the unmalted sorghum comprises sorghum with content of condensed tannins in the range of from 0.2 to 90 mg catechin equiv./g sorghum.
  • alpha amylase is selected from the group comprising SEQ ID NO:1, SEQ ID NO:5 and SEQ ID NO:9 and molecules having 60% identity to them.
  • alphaamylase has at least at least 60% identity to the sequence shown in SEQ ID NO: 1
  • alphaamylase has at least at least 60% identity to the sequence shown in SEQ ID NO: 5.
  • alphaamylase has at least at least 60% identity to the sequence shown in SEQ ID NO:9.
  • Sorghum (Sorghum bicolor (L) Moench) contains a wide variety of phenolic compounds and their levels and types depend on the genotype. Sorghums with a pigmented testa contain condensed tannins, which are the main phenolic compounds in those genotypes and are concentrated in the pigmented testa.
  • Type I sorghums do not have a pigmented testa and contain low levels of phenolic compounds and no tannins.
  • Type II and III sorghums both have a pigmented testa and contain tannins, but the tannins in type II sorghums are extracted with acidified methanol (1 % HCL) while those in type III sorghums are extracted with either methanol or acidified methanol.
  • Type II sorghums contain lower tannin levels than Type III sorghums.
  • the colour of the sorghum seed does not appear to directly correlate with the colour of the testa and thereby the amount of condensed tannin.
  • the vanillin-HCL assay involves the condensation of vanillin with monomeric flavanols and their oligomers to form a red adduct that absorbs at 500 nm.
  • the amount of condensed tannins in different sorghums was tested by the colorimetric vanillin HCL assay described in Dykes L. Tannin Analysis in Sorghum Grains. Methods Mol Biol. 2019;1931:109-120. doi: 10.1007/978-1 -4939-9039-9_8. PMID: 30652286.
  • Table 2 Tannin content of Sorghum
  • Sorghum was milled using a Lab mill 3100 and retention sieve 0.8mm (mm nominal diameter). 21g water was mixed with 7 g grist (water to grist ratio of 3 to 1). Calcium ions were added to the mash water in an amount of 85 ppm.
  • Enzyme performance was evaluated based on the final viscosities obtained at the end of the process, as well as the area of the integration of the individual viscosity measurements over the process time, from 10 mins after start to the end (See Figure 1, and Brief description of figures, Fig 1).
  • the set-back was also considered.
  • Set-back was measured as the final viscosity, minus the viscosity at the end of the holding phase after cooling to 65 °C (see Figure 1).
  • the calculated isoelectric point for a protein is a theoretical value calculated from its amino acid composition, assuming that no electrostatic interactions change the propensity for ionization.
  • the calculated pl used herein was calculated by running pepstats command from EMBOSS 6.6.0 with the Epk.dat file from 6.3.1. This pl calculation does not take predicted signal peptide, or propeptide removal into account.
  • EMBOSS 6.6.0 is available from http://emboss.sourceforge.net/ for download and installation on a LINUX computer.
  • the Epk.dat file from 6.3.1 is named: "pepstats -pkfile Epk_emboss_6_3_1.dat -filter -sequence SEQUENCEFILE", and contains:
  • the liquefaction process was monitored by in-situ viscosity measurement of the sorghum grist/water suspension using a Perten Rapid Visco Analyzer (RVA). Performance was evaluated by integrating the area underneath the viscosity curve obtained from the liquefaction process, as well as the final viscosity at the end of liquefaction process (after cooling to 65 °C, simulating a typical subsequent temperature in a process in which saccharification of the liquified starch is desirable, e.g. production of wort for beer production) and the set-back (final viscosity at end of process minus viscosity before cooling from holding temperature to final temperature).
  • RVA Perten Rapid Visco Analyzer
  • ENZ 6 and ENZ 8 comprise CBM20 domains, yet neither display the improved liquefaction performance of ENZ 1, ENZ 5 and ENZ 9.
  • ENZ 1 was particularly effective. Treatment with ENZ1 resulted in a fully liquid mash (sorghum mash after liquefaction being liquid) at a dosage of 13.3 mg enzyme protein/kg sorghum when holding temperature during the liquefaction process was 80 °, see Table 4, as well as Figure 2.
  • ENZ 4 (Amylex 6T TM, Dupont) displayed lowest final viscosity; However, the area under the viscosity curve for that enzyme (see Table 4 and FIG 2 and 3 ) indicates that it takes much longer time for this enzyme to achieve this viscosity. In contrast, the enzymes ENZ 1 , ENZ 5 and ENZ 9 achieve the lowest final viscosity, together with the lowest area under the curve of viscosity. This underscores the superior performance of these enzymes.
  • EXAMPLE 2 Evaluation of alpha-amylases liquefaction on sorghum- Set back
  • Example 1 Increase in viscosity for the samples in Example 1 (set-back) after cooling from holding (80 °C) to final (65 °C) temperature and 5 minutes rest as it is shown in Table 4, set-back and Figure 4.
  • Setback also known as starch retrogradation, is the phenomenon when starch which is not fully degraded in the liquefaction process, re-assembles to some degree after liquefaction process - this reassembly is reflected in increased viscosity on cool-down. Higher set-back means increased degree of starch retrogradation.
  • Example 2 The three best performing enzymes (ENZ 1, ENZ 5 and ENZ 9) in Example 2 also showed good performance here, i.e. they display low to no setback.
  • ENZ 1 displayed a negative set-back value.
  • Sorghum from different sources were subjected to liquefaction process as described in Materials and methods, in presence of 9 mg acid alpha-amylase ENZ 1/kg sorghum grist.
  • sorghum varieties with lower concentrations (0.38, 12.23, 28.48 and 46.38 mg catechin equivalent/g sorghum) were tested in a liquefaction process with 80 °C holding temperature.
  • ENZ 1 showed similar liquefaction performance for all sorghums, and all tested sorghums could be completely liquified when the holding temperature was set to 80 °C. See Table 2 for sorghums.
  • Table 5 Area under the curve, final viscosity and Setback The area underneath the viscosity curves as well as the final viscosities showed variation for the different sorghum mashes (see Table 5), however all sorghum mashes were completely liquified.

Abstract

La présente invention concerne un procédé de production d'un moût de brasserie qui utilise du sorgho dans la mouture.
PCT/EP2022/078390 2022-10-12 2022-10-12 Procédé de production de moût de brasserie WO2024078714A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012140075A2 (fr) 2011-04-15 2012-10-18 Novozymes A/S Procédé pour la production de moût de bière
WO2019219601A2 (fr) 2018-05-16 2019-11-21 Novozymes A/S Procédé de production de moût de bière
WO2021236715A1 (fr) * 2020-05-21 2021-11-25 Dupont Nutrition Biosciences Aps Amylases uniaxiales pour brassage avec des matériaux à teneur élevée en tanin

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012140075A2 (fr) 2011-04-15 2012-10-18 Novozymes A/S Procédé pour la production de moût de bière
WO2019219601A2 (fr) 2018-05-16 2019-11-21 Novozymes A/S Procédé de production de moût de bière
WO2021236715A1 (fr) * 2020-05-21 2021-11-25 Dupont Nutrition Biosciences Aps Amylases uniaxiales pour brassage avec des matériaux à teneur élevée en tanin

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* Cited by examiner, † Cited by third party
Title
DYKES L.: "Tannin Analysis in Sorghum Grains", METHODS MOL BIOL., vol. 1931, 2019, pages 109 - 120
MATTHEWS ET AL., JOURNAL OF INSTITUTE OF BREWING, vol. 107, no. 3, 2001, pages 185 - 194
NEEDLEMANWUNSCH, J. MOL. BIOL., vol. 48, 1970, pages 443 - 453
RICE ET AL., EMBOSS: THE EUROPEAN MOLECULAR BIOLOGY OPEN SOFTWARE SUITE, 2000
TRENDS IN GENETICS, vol. 16, pages 276 - 277
WOLFGANG KUNZE: "the Research and Teaching Institute of Brewing", 1999, article "Technology Brewing and Malting"

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