Haze-resistant beer concentrate

Abstract

The invention concerns a beer concentrate having a dry matter content of 15 to 50 wt.% and a total amino acid content of 5% to 40% by weight of dry matter, said beer concentrate comprising microbial endoprotease. Haze stability of beer concentrate is improved considerably if the concentrate is produced from a beer that has undergone yeast fermentation in the presence of endoprotease. Another aspect of the invention relates to a method of preparing a beer concentrate. The method comprises: • fermenting wort with yeast to produce a fermented wort; • separating yeast from the fermented wort to produce a beer; and • removing water from the beer to produce a beer concentrate, wherein endoprotease is added to the wort before and / or during fermentation.

Description

[0001] HAZE-RESISTANT BEER CONCENTRATE

[0002] TECHNICAL FIELD OF THE INVENTON

[0003] The invention relates to a beer concentrate that even after prolonged storage under ambient conditions, upon reconstitution with carbonated water, yields a non-hazy beer. Such a hazeresistant beer concentrate may be obtained by a process in which beer is produced by fermenting wort in the presence of endoprotease, e.g. proline-specific endoprotease, followed by removal of water from the beer.

[0004] BACKGROUND OF THE INVENTION

[0005] The popularity of domestic appliances for preparing and dispensing carbonated beverages from concentrated syrup, such as Sodastream®, has grown rapidly. These appliances produce carbonated beverages by carbonating water and mixing the carbonated water with a flavoured syrup. Given the high flexibility and convenience provided by these appliances, it would be desirable to have available beer concentrates from which beer can be produced using similar appliances.

[0006] Since beer typically contains more than 90% of water, beer can be concentrated considerably by removing most of the water. The benefits of producing beer from a concentrate have been recognized in the art. However, the production of a beer concentrate that can suitably be used to produce a good quality beer represents a challenging task.

[0007] First of all, water should be removed selectively so as to avoid loss of flavour substances, colour and / or beer components that contribute to the formation and stability of foam heads. Since the removal of water from beer favours the occurrence of chemical reactions between reactive beer components, measures need to be taken to suppress / prevent chemical reactions that give rise to perceptible changes. An example of such a perceptible change is the occurrence of turbidity in reconstituted beer concentrate after prolonged storage of the beer concentrate under ambient conditions.

[0008] Consumers expect a filtered beer to be a clear, bright, non-hazy product that remains so during its shelf life. Hazy products are often regarded as defective. Therefore, controlling haze formation is an important challenge in beer production. Beer haze can be divided into biological haze and non-biological haze. Biological haze is caused by wild bacteria or yeast due to poor hygiene during beer processing and storage. Non-biological haze is caused by the large molecular substances in beer, such as dextrins, p- glucans, proteins, and polyphenols.

[0009] According to the European Brewery Convention (EBC), the non-biological haze is classified into two types: chill and permanent. The chill haze forms when the beer is chilled to 0 °C, but re-dissolves when the beer is warmed to 20 °C or more. Permanent haze is present in beer even at 20 °C or higher temperature. It is generally believed that chill haze results from weak chemical bond (such as hydrogen bond) interaction between large molecular substances (such as proteins and polyphenols) in beer. Permanent haze is considered to be the result of strong chemical bonds (such as covalent bonds) interaction between large molecular substances. Chill haze is a precursor of permanent haze.

[0010] The stability of beer haze is strongly affected by the properties of malt. If the starch in malt is not sufficiently broken down during mashing, long-chain dextrins which cannot be used by yeast can cause turbidity. Also p-glucans and arabinoxylans may cause beer turbidity. Among the factors causing non-biological haze formation, the interaction between haze-active proteins and polyphenols is the most well-known.

[0011] An effective beer treatment with respect to haze stability is the cold storage of the beer for about seven days at -1 to -2°C. However, brewers frequently wish to accelerate the process of haze stabilization and achieve greater stability than is possible with cold storage alone.

[0012] US 4,265,920 describes a process for the concentration of aqueous alcoholic beverage solutions, containing in addition to non-volatile components, alcohol and small amounts of volatile aroma components by the selective removal of water, comprising the following steps:

[0013] (a) a first step in which substantially all of the alcohol and the more volatile aroma components are separated by a process of distillation at strongly reduced pressure, from the bulk of the aqueous solution and in which the vapors containing alcohol and more volatile aroma components obtained by said distillation process are condensed in a condenser,

[0014] (b) a second step in which the aqueous solution obtained in step (a), is concentrated by removing water in a process of freeze concentration while retaining in the solution the aroma components remaining from step a), and

[0015] (c) a third step in which the condensate containing alcohol and more volatile aroma components obtained in step (a), is mixed with the concentrate obtained in step (b ). WO 2016 / 083482 describes a method for preparing beer concentrate, comprising the steps of:

[0016] (a) subjecting beer or cider (1) to a first concentration step comprising nanofiltration (A) or reverse osmosis to obtain a retentate (2) and a fraction comprising alcohol and volatile flavour components (3), wherein the retentate (2) is characterised by the concentration of unfilterable compounds equal to or higher than 20% (w / w), as calculated from density measurement corrected for the alcohol amount;

[0017] (b) subjecting the fraction comprising alcohol and volatile flavour components (3) to a next concentration step (B) comprising freeze concentration, fractionation, preferably being distillation, or reverse osmosis, to obtain a concentrated fraction comprising alcohol and volatile flavour components (4) and a leftover fraction (5);

[0018] (c) combining (C) the retentate (2) from a) with the concentrated fraction (4) comprising alcohol and volatile flavour components from b).

[0019] US 2016 / 230133 describes a method of preparing a concentrate from an alcoholic beverage, comprising:

[0020] • subjecting an alcoholic beverage to a membrane process by which at least some water and alcohol pass through a membrane to be part of a permeate and other components of the alcoholic beverage do not pass through the membrane and are part of a retentate;

[0021] • freezing water in the retentate to form ice; and

[0022] • removing ice from the retentate to reduce water content and form a beverage concentrate having a solids concentration of at least 30% and an alcohol concentration of 20% or less.

[0023] WO 2023 / 012217 describes a process of manufacturing a liquid alcoholic beer concentrate, said process comprising:

[0024] • providing a low alcohol beer having an ethanol content of 0-1 % ABV;

[0025] • removing at least 70 wt.% of the water present in the low alcohol beer by means of membrane separation to produce a low alcohol beer concentrate, wherein the membrane separation is selected from nanofiltration, reverse osmosis and forward osmosis;

[0026] • combining the low alcohol beer concentrate with alcoholic liquid having an ethanol content of at least 30 wt.% to produce a liquid alcoholic beer concentrate having an ethanol content of 10-60 wt.%.

[0027] WO 02 / 046381 describes a method for the prevention or reduction of haze in a beverage wherein a prolyl specific endoprotease is added to the beverage.

[0028] WO 2017 / 085210 describes a process for preparing beer comprising the steps of a. preparing a mash, b. separating a wort from the mash, c. boiling of the wort, d. fermenting the wort, and e. preparing the beer, wherein a proline-specific endoprotease and a polyphenoloxidase are contacted with the mash during preparing of the mash and I or with the wort before boiling of the wort.

[0029] SUMMARY OF THE INVENTION

[0030] The inventors have found that even if beer concentrate is produced from beer that has excellent haze stability, following prolonged storage of the beer concentrate under ambient conditions, reconstitution of the beer concentrate with carbonated water yields a beer that exhibits undesirable turbidity. This haze problem increases with storage time. Furthermore, this haze does not disappear when the concentrate is combined with carbonated water to produce a reconstituted beer.

[0031] The inventors have unexpectedly discovered that the haze stability of beer concentrate can be improved considerably if the concentrate is produced from a beer that has undergone yeast fermentation in the presence of endoprotease, e.g. proline-specific endoprotease.

[0032] Accordingly, one aspect of the invention concerns a beer concentrate having a dry matter content of 15 to 50 wt.% and a total amino acid content of 5% to 40% by weight of dry matter, said beer concentrate comprising microbial endoprotease.

[0033] Another aspect of the invention relates to a method of preparing a beer concentrate. The method comprises:

[0034] • fermenting wort with yeast to produce a fermented wort;

[0035] • separating yeast from the fermented wort to produce a beer; and

[0036] • removing water from the beer to produce a beer concentrate, wherein endoprotease is added to the wort before and / or during fermentation.

[0037] DETAILED DESCRIPTION OF THE INVENTION

[0038] A first aspect of the present invention relates to a beer concentrate having a dry matter content of 15 to 50wt.% and a total amino acid content of 5% to 40% by weight of dry matter, said beer concentrate comprising microbial endoprotease. The term “beer” as used herein refers to a yeast fermented malt beverage that has optionally been hopped. Beer is commonly produced by a process that comprises the following basic steps:

[0039] • mashing a mixture comprising malted barley, optionally supplementary grains and water to produce a mash;

[0040] • separating the mash in wort and spent grains;

[0041] • boiling the wort to produce a boiled wort;

[0042] • fermenting the boiled wort with live yeast to produce a fermented wort;

[0043] • subjecting the fermented wort to one or more further process steps (e.g. maturation and filtration) to produce beer; and

[0044] • packaging the beer in a sealed container, e.g. a bottle, can or keg.

[0045] Hop or hop extract is usually added during wort boiling to impart bitterness and floral, fruity flavor notes to the final beer.

[0046] The term “beer concentrate” as used herein refers to beer from which water has been removed, e.g. by means of nanofiltration, reverse osmosis, forward osmosis and / or freeze concentration.

[0047] The term “protein” as used herein refers to polypeptides containing at least 20 amino acid residues.

[0048] The term “endoprotease” as used herein refers to a protease that is capable of hydrolysing proteins by breaking peptide bonds of nonterminal amino acids. The term “endoprotease” also encompasses inactivated forms of the enzyme, e.g. endoprotease that has been inactivated by heat treatment.

[0049] The term “proline-specific endoprotease” as used herein refers to an endoprotease that hydrolyses a protein or peptide at a position where the protein or peptide contains a prolineresidue.

[0050] The term “glutamine-specific endoprotease” as used herein refers to an endoprotease a protease that hydrolyses a protein or peptide at a position where the protein or peptide contains a glutamine-residue.

[0051] The term “amyloglucosidase” as used herein refers to an enzyme that is capable of hydrolysing terminal (1 — >4)-linked a-D-glucose residues from starch. The term “amyloglucosidase” also encompasses inactive or inactivated forms of the enzyme, e.g. amyloglucosidase that has been inactivated by heat treatment.

[0052] The term “membrane separation” as used herein refers to a separation method in which molecules are separated by passing a feed stream through a membrane that separates it into two individual streams, known as the permeate and the retentate. Examples of membrane separation include nanofiltration, reverse osmosis and forward osmosis.

[0053] The term “free amino nitrogen” as used herein refers to the combined concentration of individual amino acids and small peptides as determined by EBC method 9.10.1 - Free Amino Nitrogen in Beer by Spectrophotometry (IM).

[0054] Whenever reference is made herein to the concentration level of amino acids or the concentration level of one or more specifically mentioned amino acids, unless indicated otherwise, these concentration levels refer to combined concentration of free amino acids and amino acid residues. It will be understood that these concentration levels may be determined by fully hydrolysing the peptides and proteins that are contained in a beer concentrate before carrying out a quantitative amino acid analysis by means of HPLC.

[0055] The presence of microbial endoprotease in a beer concentrate may suitably be determined by proteome characterisation, notably by bottom-up proteomics. In bottom-up proteomics analyses, protein is isolated and enzymatically or chemically cleaved into peptides. The resultant complex peptide mixture is fractionated using chromatography, e.g. reversed phase chromatographic. The peptides eluting from the chromatographic column are ionized by electrospray ionization (ESI) and analyzed by MS. The power of MS lies not only in its parts per million (ppm) mass measurement accuracy, but in the ability to perform tandem MS (MS / MS) measurements that provide additional information specific for the peptide amino acid sequence. Typical LC MS / MS involves the acquisition of a preliminary mass spectrum (MS1) of the intact (precursor) peptide, dissociation of the isolated precursor ion of interest into smaller fragments, and subsequent mass analysis of the fragments (MS2). The process is repeated for the duration of the LC separation of the peptide mixture. Peptide fragmentation typically results from collision-induced dissociation (CID), or alternative techniques such as electron capture dissociation (ECD) or electron transfer dissociation (ETD).

[0056] The presence of active microbial endoprotease in a beer concentrate may be determined by using an enzyme assay that is capable of measuring endoprotease activity. A suitable assay for measuring activity of proline-specific endoprotease is described in WO 2007 / 101888. The term “iso-alpha acids” as used herein refers to substances selected from the group of isohumulone, isoadhumulone, isocohumulone, pre-isohumulone, post-isohumulone and combinations thereof. The term “iso-alpha acids” encompasses different stereo-isomers (cis- iso-alpha acids and trans-iso-alpha acids). Iso-alpha acids are typically produced in beer from the addition of hops to the boiling wort. They may also be introduced into the beer in the form of pre-isomerised hop extract.

[0057] The term “amino acid sequence” is synonymous with the terms “polypeptide,” “protein,” and “peptide,” and are used interchangeably. Where such amino acid sequences exhibit activity, they may be referred to as an “enzyme.” The conventional one-letter or three-letter codes for amino acid residues are used, with amino acid sequences being presented in the standard amino-to-carboxy terminal orientation (i.e., N^C).

[0058] The term “nucleic acid” encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding a polypeptide. Nucleic acids may be single stranded or double stranded, and may be chemical modifications. The terms “nucleic acid” and “polynucleotide” are used interchangeably. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and the present compositions and methods encompass nucleotide sequences that encode a particular amino acid sequence. Unless otherwise indicated, nucleic acid sequences are presented in 5'-to-3' orientation.

[0059] As used herein, “percent sequence identity” means that a particular sequence has at least a certain percentage of amino acid residues identical to those in a specified reference sequence, when aligned using the CLUSTAL W algorithm with default parameters. See Thompson et al. (1994) Nucleic Acids Res. 22:4673-4680. Default parameters for the CLUSTAL W algorithm are:

[0060] Gap opening penalty: 10.0

[0061] Gap extension penalty: 0.05

[0062] Protein weight matrix: BLOSUM series

[0063] DNA weight matrix: IUB

[0064] Delay divergent sequences %: 40

[0065] Gap separation distance: 8

[0066] DNA transitions weight: 0.50

[0067] List hydrophilic residues: GPSNDQEKR

[0068] Use negative matrix: OFF

[0069] Toggle Residue specific penalties: ON

[0070] Toggle hydrophilic penalties: ON Toggle end gap separation penalty: OFF.

[0071] Deletions are counted as non-identical residues, compared to a reference sequence. Deletions occurring at either terminus are included.

[0072] The beer concentrate of the present invention preferably is a concentrate of a lager beer.

[0073] According to a further preferred embodiment, the beer concentrate is a concentrate of a 100% malt beer.

[0074] Preferably, the beer concentrate of the invention has an ethanol content of 12-50 wt.%. More preferably, the beer concentrate of the invention has an ethanol content of 14-40 wt.%. Even more preferably, the beer concentrate of the invention has an ethanol content of 15-35 wt.%.

[0075] The beer concentrate preferably has a dry matter content of 18 to 45 wt.%, more preferably of 20 to 42 wt.% and most preferably of 22 to 40 wt.%.

[0076] The total amino acid content of the beer concentrate is preferably in the range of 6% to 33% by weight of dry matter, more preferably 7% to 25% by weight of dry matter and most preferably 8% to 20% by weight of dry matter.

[0077] According to another embodiment of the invention the beer concentrate has a free amino nitrogen (FAN) content of 0.2-2.0 milligram per gram of dry matter. More preferably, the beer concentrate has a FAN content of 0.3-1.8 milligram per gram of dry matter. Even more preferably, the beer concentrate has a FAN content of 0.4-1 .6 milligram per gram of dry matter.

[0078] According to a preferred embodiment, the microbial endoprotease is selected from microbial proline-specific endoprotease, microbial glutamine-specific endoprotease and combinations thereof. Most preferably, the microbial endoprotease is microbial proline-specific endoprotease.

[0079] Preferably, at least a part of the microbial endoprotease in the beer concentrate is still active, i.e. capable of hydrolysing proteins by breaking peptide bonds of nonterminal amino acids.

[0080] The microbial endoprotease employed in accordance with the present invention preferably is a fungal endoprotease, more preferably the microbial endoprotease is derived from Aspergillus, most preferably from Aspergillus niger. In a preferred embodiment, the microbial endoprotease that is employed in accordance with the present invention is a polypeptide having proline-specific endoprotease activity, selected from the group consisting of:

[0081] (a) a polypeptide which has an amino acid sequence which has at least 80%, more preferably at least 90%, even more preferably at least 95% and most preferably at least 97% overall amino acid sequence identity with SEQ ID NO: 1 , SEQ ID NO : 2, OR SEQ ID NO: 3 or a fragment thereof;

[0082] (b) a polypeptide which is encoded by a polynucleotide which hybridizes under low stringency conditions with (i) the nucleic acid sequence of SEQ ID NO : 4, SEQ ID NO: 5, SEQ ID NO : 6 OR SEQ ID NO: 7 or a fragment thereof which is at least 80%, preferably at least 90% identical over 60, preferably over 100 nucleotides, more preferably at least 90% identical over 200 nucleotides, or (ii) a nucleic acid sequence complementary to the nucleic acid sequence of (i). The relevant sequence listings are disclosed in WO 02 / 46381 as SEQ ID 4, 5, 7, 1 , 2, 3 and 6 respectively.

[0083] In another preferred embodiment, the microbial endoprotease that is employed in accordance with the present invention is a polypeptide having glutamine-specific endoprotease activity, said endoprotease having at least 75, 80, 85, 90, 95, 98, 99 or 100% sequence identity to SEQ ID NO:8, SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 or SEQ ID NO: 12 or an endoprotease active fragment thereof such as a mature protein. The relevant sequence listings are disclosed in WO 2022 / 266456 as SEQ ID 1 , 2, 3, 4 and 16.

[0084] The active form of the microbial endoprotease present in the beer concentrate preferably hydrolyses a peptide bond at the carboxy-terminal end of proline residues or glutamine residues. Most preferably, the endoprotease hydrolyses a peptide bond at the carboxy-terminal end of proline residues.

[0085] The beer concentrate of the present invention preferably contains microbial amyloglucosidase. Preferably, at least a part of the amyloglucosidase is present as active amyloglucosidase.

[0086] Preferably, the concentrate according to the invention contains 30% to 95% of carbohydrates by weight of dry matter. More preferably, the concentrate contains 50% to 92% of carbohydrates by weight of dry matter. Even more preferably, the concentrate contains 70% Preferably, starch hydrolysis products having a degree of polymerization of 3 or more constitute at least 50 wt.%, more preferably at least 70 wt.% and most preferably at least 80 wt.% of the carbohydrates in the beer concentrate.

[0087] Preferably, the concentrate contains 0.2 mg to 4.0 mg of iso-alpha acids per gram of dry matter. More preferably, the concentrate contains 0.3 mg to 3.0 mg of iso-alpha acids per gram of dry matter. Even more preferably, the concentrate contains 0.4 mg to 2.5 mg of iso-alpha acids per gram of dry matter.

[0088] In a particularly preferred embodiment, the bulk of the protein in the beer concentrate originates from cereal. Accordingly, preferably at least 80 wt.%, more preferably at least 90 wt.% of the protein in the beer concentrate is cereal protein. The cereal protein in the beer concentrate preferably originates from barley, wheat, sorghum, rye, oats, maize, rice or a combination thereof.

[0089] The proteins originating from these cereals typically contain relatively high levels of proline. Accordingly, in a preferred embodiment, proline (free and bound) represents 20-60 wt.% of the total amount of amino acids that is contained in the beer concentrate. More preferably, proline represents 30-50 wt.% of the total amount of amino acids that is contained in the beer concentrate.

[0090] Riboflavin, free fatty acids (e.g. linoleic acid), amino acids and small peptides are substances that are naturally present in malted barley and that typically occur in significant concentrations in beer. The beer concentrate of the present invention is preferably obtained from beer using a concentration method that only removes water, or only water and low molecular weight substances and ions. As a result, the beer concentrate typically contains appreciable levels of riboflavin.

[0091] The riboflavin content of the beer concentrate is preferably in the range of 2-30 microgram per gram of dry matter, more preferably 3-20 microgram per gram of dry matter and most preferably 4-12 microgram per gram of dry matter.

[0092] Preferably, reconstitution of one part by weight of the beer concentrate with 4 parts by weight of demineralized water 6 months after production of the beer concentrate yields a beer having a turbidity (at 90° angle) of 0 to 3.0 EBC units, more preferably of 0 to 2.0 EBC units and most preferably of 0.1 to 1.0 EBC units. Another aspect of the invention relates to a method of preparing a beer concentrate, said method comprising:

[0093] • fermenting wort with yeast to produce a fermented wort;

[0094] • separating yeast from the fermented wort to produce a beer; and

[0095] • removing water from the beer to produce a beer concentrate; wherein endoprotease is added to the wort before and / or during fermentation.

[0096] The beer obtained after separation of the yeast preferably has an ethanol content of 0-12% ABV, more preferably of 2-10% ABV and most preferably of 3-8% ABV.

[0097] Prior to water removal, the beer preferably has a turbidity (at 90° angle) of 0 to 1.0 EBC units, more preferably of 0 to 0.8 EBC units and most preferably of 0.1 to 0.6 EBC units.

[0098] The beer concentrate that is produced in the present method preferably has a dry matter content of 2 to 10 wt.%, more preferably of 3 to 8 wt.% and most preferably of 4 to 6 wt.%.

[0099] The beer concentrate that is produced by the present method preferably has a total amino acid content of 5% to 40% by weight of dry matter, more preferably a total amino acid content of 6% to 33% by weight of dry matter, even more preferably a total amino acid content of 7% to 25% by weight of dry matter and most preferably a total amino acid content of 8% to 20% by weight of dry matter.

[0100] The beer concentrate that is produced in the present method preferably has an ethanol content of 12-50 wt.%, more preferably an ethanol content of 14-40 wt.% and most preferably an ethanol content of 15-35 wt.%.

[0101] The beer concentrate that is obtained by the present method preferably is a beer concentrate as described herein before.

[0102] Preferably, the wort used in this method is produced by:

[0103] • mashing an aqueous liquid comprising malt and water to produce mash; and

[0104] • separating the mash into wort and spent grain; wherein amyloglucosidase is added to the aqueous liquid before or during the mashing. The inventors have found that the application of amyloglucosidase during mashing further improves the haze stability of the beer concentrate. According to a particularly preferred embodiment, amyloglucosidase is present in the aqueous liquid during at least 10 minutes of the mashing, more preferably during at least 20 minutes of the mashing and most preferably during at least 30 minutes of the mashing.

[0105] The mashing of the aqueous liquid comprising malt and water preferably comprises heating the aqueous liquid to a temperature of at least 60°C, more preferably to a temperature of 65- 90°C, most preferably to a temperature of 70-85°C for at least 10 minutes, preferably for 30- 100 minutes.

[0106] Amyloglucosidase is preferably added to the mash to provide at least 0.1 unit of amyloglucosidase activity per gram of starch, more preferably to provide 0.2-12 units of amyloglucosidase activity per gram of starch and most preferably to provide 0.3-6 units of amyloglucosidase activity per gram of starch. One unit of amyloglucosidase activity is defined as the amount of enzyme which hydrolyzes 1 micromole maltose per minute under the standard conditions: 37°C, pH 4.3, substrate: maltose 100 mM, buffer: acetate 0.1 M, reaction time 6 minutes. Examples of suitable amyloglucosidases are described in WO 2012 / 140075.

[0107] According to another embodiment of the invention the fermentation is carried out at temperature of 0 to 24 °C, preferably at temperature of 2 to 17 °C, more preferably at temperature of 6 to 15 °C.

[0108] The duration of the fermentation preferably exceeds 8 hours, more preferably the duration is in the range of 1-40 days, more preferably of 4-30 days and most preferably of 6-25 days.

[0109] The microbial endoprotease employed in the present method preferably is a fungal endoprotease, more preferably the microbial endoprotease is derived from Aspergillus, most preferably from Aspergillus niger.

[0110] The endoprotease employed in the present method is preferably selected from proline-specific endoprotease, glutamine-specific endoprotease and combinations thereof. Examples of suitable proline-specific endoproteases are described in WO 2002 / 046381. Examples of suitable glutamine-specific proteases are disclosed in WO 2022 / 266456. Most preferably, the endoprotease employed is proline-specific endoprotease.

[0111] Proline-specific endoprotease is preferably added to the wort to provide at least 0.01 units of endoproteose activity per litre, more preferably to provide 0.03-2 units of endoprotease activity per litre and most preferably to provide 0.05-1 units of endoprotease activity per litre. One unit of proline-specific endoprotease activity being defined as the quantity of enzyme that liberates 1 pmol of pnitroanilide per minute under the test conditions described in W02007 / 101888.

[0112] The endoprotease employed in the present method preferably hydrolyses a peptide bond at the carboxy-terminal end of proline residues of glutamine residues. Most preferably, the endoprotease hydrolyses a peptide bond at the carboxy-terminal end of proline residues.

[0113] Preferably, the endoprotease is present during at least 12 hours of the fermentation, more preferably during at least 1 day of the fermentation and most preferably at least 3 days of the fermentation.

[0114] Water can suitably be removed from the beer through freeze concentration. In this method, water is withdrawn from the beer by the phase transformation from liquid to ice crystal. This process has mainly three stages: crystallization of water, growth of water crystals and separation of water crystals, performed in specially designed equipment for each purpose. For instance, scraped-surface heat exchanger, growth recrystallizer and separation wash column, respectively. Basically, the temperature of the alcohol-free beer is reduced to a value such as to freeze at least a part of its water without reaching the eutectic point of the mixture. When the ice crystals are sufficiently large, e.g. not smaller than 100 pm in diameter, said crystals can be separated from the concentrated liquid for example using wash-columns. Because of the low process temperature, lower than 0 °C, thermal degradation and aroma losses by evaporation are avoided.

[0115] Water may also be removed from the beer by means of membrane separation using a membrane with a magnesium sulphate rejection of 80-100%, more preferably 90-100% and most preferably 95-100% when measurement is carried out using 2,000 mg / L aqueous magnesium sulphate solution at 0.48 MPa, 25°C and 15% recovery.

[0116] According to a particularly preferred embodiment, water is removed from the beer by means of reverse osmosis or forward osmosis using a membrane with a sodium chloride rejection of 80-100%, more preferably 90-100% and most preferably 95-100% when measurement is carried out using 2000 mg / L sodium chloride solution at 10.3 bar, 25°C, pH 8 and 15% recovery.

[0117] The invention is further illustrated by the following non-limiting examples. EXAMPLES

[0118] Comparative Example A

[0119] A lager beer (Beer A) was produced as follows. An aqueous malt mixture comprising water and barley malt was prepared by mashing a mixture of malt and water to produce a mash (mashing scheme: 15 minutes at 60°C; 60 minutes at 65°C; 8 minutes at 78°C). The mash was separated in wort and spent grain; then the wort was boiled in the presence of hops to produce a boiled wort.

[0120] The wort so obtained (specific gravity = 15 Plato) was fermented for 23 days at a temperature between 9°C and 11°C with live yeast to produce a fermented wort. The fermented wort was then subjected to maturation and filtration to produce a lager. The lager had an alcohol content of 7% ABV and a turbidity (at 90° angle) of 0.60 EBC units.

[0121] The lager beer was concentrated to 20% ABV by means of freeze concentration, yielding Beer Concentrate A.

[0122] Example 1

[0123] A lager beer (Beer 1) was produced in the same way as in Comparative Example A, except that proline-specific endoprotease (Brewers Clarex® 3 g / hL, obtainable from DSM Food Specialties, Delft, Netherlands) was added during fermentation.

[0124] Beer 1 was concentrated by freeze concentration as described in Comparative Example A to yield Beer Concentrate 1.

[0125] Example 2

[0126] A lager beer (Beer 2) was produced in the same way as in Example 1 , except that additionally amyloglucosidase (Amigase Mega L, obtainable from DSM Food Specialties, Delft, Netherlands) was added during the mashing (5 kg / 1000 kg) at 20 minutes at 65°C.

[0127] Beer 2 was concentrated by freeze concentration as described in Comparative Example A to yield Beer Concentrate 2.

[0128] Example 3

[0129] Beer concentrates A, 1 and 2 were reconstituted at t = 0 and after 3 months storage at 30 °C by adding carbonated water to the concentrates to a final ABV of 5%. Turbidity of the reconstituted beers was measured at a scattering angle of 90°, at a temperature of 0°C, and at a wavelength of 650 nm. Results are shown in Table 1.

[0130] Table 1

Claims

CLAIMS1. A beer concentrate having a dry matter content of 15 to 50 wt.% and a total amino acid content of 5% to 40% by weight of dry matter, said beer concentrate comprising microbial endoprotease.

2. Beer concentrate according to claim 1 , wherein the beer concentrate has an ethanol content of 12-50 wt.%.

3. Beer concentrate according to claim 1 or 2, wherein the beer concentrate contains 30-90% of carbohydrates by weight of dry matter.

4. Beer concentrate according to any one of the preceding claims, wherein the beer concentrate has a free amino nitrogen (FAN) content of 0.2 to 2.0 milligram per gram of dry matter.

5. Beer concentrate according to any one of the preceding claims, wherein the microbial endoprotease is a microbial proline-specific endoprotease.

6. Beer concentrate according to any one of the preceding claims, wherein the microbial endoprotease is a fungal endoprotease.

7. Beer concentrate according to claim 6, wherein the fungal endoprotease is derived from Aspergillus niger.

8. Beer concentrate according to any one of the preceding claims, wherein proline (free and bound) represents 20-60 wt.% of the total amount of amino acids that is contained in the beer concentrate.

9. A method of preparing a beer concentrate, said method comprising:• fermenting wort with yeast to produce a fermented wort;• separating yeast from the fermented wort to produce a beer; and• removing water from the beer to produce a beer concentrate; wherein endoprotease is added to the wort before and / or during fermentation.

10. Method according to claim 9, wherein the endoprotease is present during at least 12 hours of the fermentation.

11. Method according to claim 9 or 10, wherein the endoprotease is a proline-specific endoprotease.

12. Method according to claim 11 , wherein the proline-specific endoprotease is added to the wort to provide at least 0.01 units of endoproteose activity per litre, wherein one unit of proline-specific endoprotease activity is defined as the quantity of enzyme that liberates 1 pmol of p-nitroanilide per minute under the test conditions described in W02007 / 101888.

13. Method according to any one of claims 9-12, wherein the wort is produced by:• mashing an aqueous liquid comprising malt and water to produce mash; and• separating the mash into wort and spent grain; wherein amyloglucosidase is wherein amyloglucosidase is added to the aqueous liquid before or during the mashing.

14. Method according to claim 13, wherein the amyloglucosidase is preferably added to the mash to provide at least 0.1 unit of amyloglucosidase activity per gram of starch, wherein one unit of amyloglucosidase activity is defined as the amount of enzyme which hydrolyzes 1 micromole maltose per minute under the standard conditions: 37°C, pH 4.3, substrate: maltose 100 mM, buffer: acetate 0.1 M, reaction time 6 minutes.

15. Method according to any one of claims 9-14, wherein the method yields a beer concentrate according to any one of claims 1-8.

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