Foam stability

Abstract

The present invention is directed to beer with reduced levels of AcHFA. It has been found that beer with reduced levels of AcHFA has improved foam stability. The present invention provides beer with reduced levels of AcHFA and methods for removing AcHFA from beer or precursors of AcHFA from wort during or after fermentation.

Description

[Technical field]

[0001] The present invention is directed to beer with improved foam stability and to a method for improving the foam stability of beer. [Background technology]

[0002] Beer is one of the most popular alcoholic beverages in the world. Beer is prepared by fermentation of a sugar-containing aqueous matrix derived from grains using yeast, which converts the sugar into ethanol ("alcohol"). The process of producing beer is generally known, and a person skilled in the art can obtain beer based on common general knowledge (see, for example, The Brewers Handbook (2nd Edition) of Ted Goldammer (2008, Apex publishers)) and the information disclosed herein.

[0003] Beer is typically made from grains such as barley, although other grain types can be used, including but not limited to wheat or sorghum. Beer is typically produced by a process that includes the following basic steps: mashing a mixture of grains and water to produce a mash; separating the mash into wort and spent grains; boiling the wort to produce boiled wort; fermenting the boiled wort with live yeast (such as Saccharomyces pastorianus or Saccharomyces cerevisiae) to produce fermented wort; subjecting the fermented wort to one or more additional process steps (e.g., maturation and filtration) to produce beer; and packaging the beer in sealed containers, such as bottles, cans, or barrels.

[0004] In an exemplary process for producing barley malt beer, barley is malted, which means that it is germinated and then dried ("roasted") to produce malt. This process is important for the formation of taste and color compounds, as well as enzymes that are important for other flavor development and starch degradation. The malt is then milled and suspended in water ("mashed"). The mash is heated to promote starch degradation. Filtration then results in the wort, which is an almost clear aqueous solution of fermentable sugars that also contains various flavors and aromas, as well as many other compounds. Both desirable and undesirable flavor compounds are present in the wort.

[0005] The wort is boiled to sterilize, precipitate proteins, and concentrate. Hops are optionally added to impart bitterness and flavor. This mixture is subjected to fermentation after removal of the sediment. Fermentation converts fermentable sugars into ethanol and carbon dioxide, and also forms a variety of novel flavor compounds. At the same time, the yeast used in fermentation brings about many other chemical transformations. To optimize appearance and taste, the beer can be filtered and / or stored after fermentation.

[0006] An important aspect of beer is the head of the beer. The head is the foam layer on top of the beer. This foam comes from carbon dioxide, which is present in the beer due to fermentation and / or post-fermentation additions, but which is essentially dissolved in the beer as a result of the high pressure in the beer container (e.g., can or bottle). Releasing the beer from the container, for example into a glass, causes the formation of carbon dioxide bubbles, which rise through the beer liquid to the top of the glass and form the foam.

[0007] A notable feature of beer foam is its stability. In contrast to other foaming yeast-fermented beverages (e.g. champagne), beer foam is stable. This is caused by (among other things) proteins and isomerized hop acids in the beer, which take their place at the bubble-liquid interface as the carbon dioxide bubbles form and rise through the beer. Once they reach the top of the liquid, these components stabilize the beer foam bubbles. As a result, the foam layer remains intact for long periods of time. An intact and stable foam layer is considered an important aspect of an enjoyable beer.

[0008] However, foam stabilization is time-limited. Within a few minutes after pouring, the foam layer becomes thinner and eventually no foam remains at all. It can happen that this process occurs faster than the consumer can consume the beer. This means that the beer becomes less enjoyable over time due to the disappearing foam layer. For slow drinkers, the foam may disappear before they finish the beer.

[0009] The present invention provides beer with improved foam stability and a method for improving the foam stability of beer. Summary of the Invention

[0010] The present invention is directed to beer with reduced levels of AcHFA. It has been found that beer with reduced levels of AcHFA has improved foam stability. The present invention provides beer with reduced levels of AcHFA and a method for removing AcHFA from beer or precursors of AcHFA from wort during or after fermentation. [Brief description of the drawings]

[0011] [Figure 1] FIG. 1 shows a comparison of foam stability before and after the adsorption process using methylcellulose ester adsorbents. [Figure 2a] FIG. 1 shows the effect of AcHFA addition on foam stability of hopped beer. [Figure 2b] FIG. 1 shows the effect of AcHFA addition on foam stability of unhopped beer. [Figure 3a] FIG. 1 shows the effect of AcHFA removal from hopped beer. [Figure 3b] FIG. 1 shows the effect of AcHFA removal from unhopped beer. [Figure 4] FIG. 1 shows AcHFA removal from beer by various adsorbents. [Diagram 5] FIG. 1 shows the activity of two adsorbents in removing AcHFA from beer. [Figure 6] FIG. 1 shows the installation of an adsorption filter in an industrial scale beer brewing process. [Figure 7a] FIG. 1 shows ATF-1 mutations in yeast. [Figure 7b] FIG. 1 shows ethanol production in ATF-1-deficient yeast (strain a), ATF-2-deficient yeast (strain b), and yeast lacking both ATF-1 and ATF-2 (strain c) compared to unmodified yeast (WT). [Figure 8a] FIG. 1 shows an exemplary configuration suitable for plant scale for removing AcHFA from beer. [Figure 8b] FIG. 1 shows an exemplary configuration suitable for plant scale for removing AcHFA from beer. [Figure 9] FIG. 1 shows increased foam stability in beer produced using factory appropriate scale. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The present invention provides a beer containing less than 2 mg / l AcHFA, the AcHFA being a C12-C22 fatty acid containing a carboxylic acid group and a C11-C21 linear alkyl group, the alkyl group being optionally partially unsaturated and substituted with at least one hydroxyl group and at least one acetate group. Preferably, the beer contains less than 1.5 mg / l, preferably less than 1.0 mg / l, more preferably less than 0.5 mg / l, even more preferably less than 0.25 mg / l, even more preferably less than 0.1 mg / l AcHFA. Thus, the present invention relates to a beer containing less than 10 mg / l AcHFA, such as less than 9 mg / l, less than 8 mg / l, less than 7 mg / l, less than 6 mg / l, less than 5 mg / l, less than 4 mg / l, or less than 3 mg / l.

[0013] It has now been found that AcHFA is formed by the action of acetyltransferase enzymes (ATFs) during normal fermentation of wort by yeast. It has been found that AcHFA is usually present in the beer obtained by fermentation at a level of at least 2 mg / l, sometimes at least 3 mg / l, or even at least 4 mg / l, or at least 5 mg / l, or at least 6 mg / l, or at least 7 mg / l, or at least 8 mg / l, or at least 9 mg / l, or at least 10 mg / l. Thus, AcHFA can be present in the beer obtained by fermentation in an amount higher than 1 mg / l, certainly higher than 0.5 mg / l, and most certainly higher than 0.25 mg / l. The inventors have found that AcHFA is a negative factor for foam and has a negative effect on foam stability. By modifying the fermentation process so that the amount of AcHFA in the final beer is reduced, the foam stability can be increased by at least 10% and up to 40%.

[0014] AcHFAs ("acetylated hydroxy fatty acids") are C12 to C22 fatty acids containing a carboxylic acid group and a C11 to C21 linear alkyl group, which may be partially unsaturated and is substituted with at least one hydroxy group and at least one acetate group. 3 CCO 2 -) is abbreviated (as is common in the art) as -OAc.

[0015] AcHFA can be defined as structure 1 [ka] [In Structure 1, n=an integer of 4 to 9, [ka] , A, B, C and / or D may each be the same or different; a) [ka] is a single bond, in which case: One of A and B is H, OH or OAc, and the other of A and B is H; one of C and D is H, OH or OAc and the other of C and D is H; or b) [ka] is a double bond, in which case, One of A and B is H, and the other of A and B does not exist (which means that the other of A and B does not exist), and one of C and D is H, and the other of C and D does not exist (which means that the other of A and B does not exist); provided that in structure 1, at least one of every A, B, C, and D is OH, and at least one of every A, B, C, and D is OAc.

[0016] As is commonly known, the double bond can be in the cis or trans configuration, although the cis configuration is preferred. Furthermore, as is common in organic acids, the acid group can be in the neutral form (-CO 2 H), but can also take the ionic form (-CO 2 - ), or in the form of a salt ((-CO 2 ) x M, where M may be any metal ion, preferably a metal ion available in beer, such as, for example, Na, K, Ca, Mg, Fe, Cu, Zn or Mn ions, where x=1 when M is monovalent (Na or K), and x may be 1, 2 or 3 for higher valence ions. The carbon atoms bearing OH or OAc can independently have the R or S configuration, but preferably the adjacent carbon atoms bearing OH and OAc groups both have the R configuration or both have the S configuration (RR and SS). Alternatively, one carbon atom of the adjacent carbon atoms bearing OH and OAc groups has the S configuration and the other of the two adjacent carbon atoms has the R configuration (RS or SR).

[0017] Preferably, the AcHFA contains one hydroxy group and one acetate group, optionally among a plurality of hydroxy and / or acetate groups, located on adjacent carbon atoms. More preferably, the AcHFA is a C16-C20 fatty acid (in structure 1, n=6-8), most preferably a C18 fatty acid (in structure 1, n=7). It is much preferred if the AcHFA contains one or two double bonds, preferably one double bond. The double bond is preferably located on the 6th, 9th, 12th or 15th carbon atom counting from the carboxylic acid group. Most preferably, the double bond is located on the 9th carbon atom.

[0018] In a much more preferred embodiment, AcHFA is represented by structure 2: [ka] [In structure 2, n=1, 2, or 3, preferably 2 or 3, most preferably 3; m=1 or 2, preferably 2; One of A and B is OH and the other of A and B is OAc.

[0019] For structure 2, the double bonds may be in either the cis or trans configuration (a line format indicating that the carbon-carbon single bonds extending from the carbon-carbon double bond may be oriented in any direction). [ka] The acid group in Structure 2 can be in the neutral form as depicted, but can also be in the ionic or salt form as defined above.

[0020] In a much more preferred embodiment, AcHFA is represented by structure 3: [ka] [In structure 3, one of A and B is OH and the other of A and B is OAc.] In these embodiments, AcHFA is (cis or trans; RR, SS, RS or SR) 12-acetoxy-13-hydroxyoctadec-9-enoic acid (3a) or (cis or trans; RR, SS, RS or SR) 13-acetoxy-12-hydroxyoctadec-9-enoic acid (3b). [ka] [ka]

[0021] AcHFAs have been found to occur naturally in beer, as they are formed during fermentation by the yeast enzyme acetyltransferase (ATF).

[0022] An AcHFA precursor is a dihydroxy fatty acid, such as structures 1-3 defined above, that has an OH-group at each position where an OAc-group is located in AcHFA. The yeast ATF enzyme acetylates (at least) one of the OH-groups to convert the AcHFA precursor to AcHFA.

[0023] Additionally, beers with reduced AcHFA content have been found to have improved foaming properties compared to similar regular beers. In particular, reduced amounts of AcHFA increase the foam stability of the beer.

[0024] In this specification, the foam stability of beer is measured based on the standard established by the NIBEM Institute and is expressed in seconds ([s]). Therefore, the foam stability of beer indicates the time in seconds during which the foam layer is stable under standard test conditions.

[0025] The NIBEM method is commonly known and can be found as the EBC method in Analysis 9.42.1. The test is performed at 20°C and atmospheric pressure. Foam stability is measured as the time elapsed from the time the foam is prepared under reference conditions (including the period during which the foam is poured) until the height of the foam layer has decreased by 3 cm.

[0026] Beer may refer to any type of beer, including, but not limited to, ale, porter, stout, lager and bock beer. The beer is preferably a malt-based beer, i.e., a beer prepared by fermentation of wort prepared from malt. Preferably, the beer is a lager beer, which is a beer obtained by fermenting at 7-15°C using bottom-fermenting yeast, followed by maturation in storage at low temperature. Lager beers include, for example, pilsner. Most preferably, the beer described herein is a pilsner. A pilsner is a pale lager beer.

[0027] Beer is understood in the broad sense herein to include both regular (alcohol-containing) and low or zero alcohol ("NA") beers. Beer in the present specification is therefore preferably beer with an ethanol content of 0-15% by volume ("ABV"), preferably 1-15% by volume. Beer may be fortified with hops ("hopped beer") or may not have added hops ("unhopped beer"). Hopped beers include beers prepared using modified hops such as ρ-hops.

[0028] In a preferred embodiment, the beer is a regular beer. In this context, "regular beer" is a normal brewed beer obtained using a fermentation process that results in ethanol of more than 1% by volume. Thus, regular beer as defined herein has an ethanol content of more than 1% by volume, preferably less than 15% by volume. The ethanol content of regular beer is preferably 2-15% by volume, more preferably 2.5-12% by volume, more preferably 3.5-9% by volume. The regular beer is preferably a lager beer as described above, most preferably a pilsner. The skilled person can obtain regular beer, among which regular lager beer and pilsner, for example by the methods described in The Brewers Handbook (2nd edition) of Ted Goldammer (2008, Apex publishers) or the methods disclosed herein. Alternatively, regular beer can be obtained commercially.

[0029] In another preferred embodiment, the beer is a zero or low alcohol beer ("NA beer"). In the present context, a "zero or low alcohol beer" is a beer having an ethanol content of 1.0% by volume ("ABV") or less, preferably 0.5% by volume or less, more preferably 0.2% by volume or less. Thus, an NA beer is a beer having an ethanol content of 0-1.0% by volume, preferably 0-0.5% by volume, etc.

[0030] NA beer can be obtained, for example, by dealcoholization of regular beer ("dealcoholized beer") or by limited ethanol fermentation of wort ("limited fermentation beer").

[0031] One way to obtain NA beer as defined herein is to subject a conventionally brewed beer to a dealcoholization step, such as a rectification step, a reverse osmosis step, a dialysis step or a freeze concentration step, in order to remove ethanol from the fermented beer. These techniques are described, for example, in Branyik et al., J. Food. Eng. 108 (2012) 493-506, or Mangindaan et al., Trends in Food Science&Technology 71 (2018) 36-45.

[0032] Another way to obtain NA beer is to make the beer by a limited fermentation process, resulting in a limited fermentation beer, which is another type of NA beer as defined herein.

[0033] Limited fermentation beer is defined as fermented beer obtained by limited ethanol fermentation of wort. Limited ethanol fermentation of wort is a fermentation with less net ethanol formation, i.e. limited fermentation as defined herein results in ethanol of 1% or less by volume, preferably 0.5% or less by volume, more preferably 0.2% or less by volume. Thus, limited fermentation beer has an ethanol content of 1.0% or less by volume, preferably 0.5% or less by volume, more preferably 0.2% or less by volume.

[0034] Limited fermentation of wort is a process in which the ethanol content of the product directly obtained from fermentation is 1.0% by volume or less, preferably 0.5% by volume or less, more preferably 0.2% by volume or less. Those skilled in the art are aware of various limited fermentation techniques in which the net ethanol formation is not significant. An example is limited ethanol fermentation of wort, characterized by: at a temperature below 7°C, preferably between -1 and 4°C, for example between -0.5 and 2.5°C, preferably for 8 to 72 hours, more preferably for 12 to 48 hours ("low-temperature contact-fermented beer"); and / or a short (e.g. less than 2 hours) fermentation time, in which the fermentation is stopped rapidly by temperature inactivation, e.g. rapid cooling to -0.5 to 1°C, optionally followed by pasteurization ("stopped fermentation beer"); and / or Fermentation with a yeast strain that produces low amounts of ethanol under the applied fermentation conditions, for example a yeast strain that produces less than 0.2 g ethanol per gram fermentable sugar in the wort, preferably less than 0.1 g ethanol per gram fermentable sugar. Suitable strains (e.g. Crabtree-negative strains) are known in the art and the amount of ethanol produced under varying fermentation conditions can be determined by routine experimentation ("yeast-limited beer"); and / or fermentation using a first ethanol-producing yeast strain in the presence of an ethanol-consuming second yeast strain, such as Saccharomyces rouxii, in an amount sufficient to consume substantially all of the ethanol produced by the first yeast strain; and / or wort with a content of fermentable sugars which, after completion of the fermentation, results in a maximum yield of 1.0% by volume of alcohol, in which case the wort generally has a content of fermentable sugars of less than 17.5 g / l, preferably less than 12 g / l, more preferably less than 8 g / l ("sugar-deficient wort beer").

[0035] The limited fermentation beer has not been subjected to a dealcoholization step to achieve said ethanol content of 1.0% by volume or less, preferably 0.5% by volume or less, more preferably 0.2% by volume or less. The skilled person is aware of various techniques suitable for dealcoholization of fermented beer (see Branyik et al. and Mangindaan et al. above), but these techniques have not been applied to achieve said ethanol content. However, the limited fermentation beer herein can be optionally subjected to a dealcoholization step to reduce the ethanol content resulting from fermentation from said 1.0% by volume or less, preferably 0.5% by volume or less, more preferably 0.2% by volume or less to an ethanol content further reduced. However, preferably no dealcoholization step is performed at all for the limited fermentation beer defined herein.

[0036] The limited fermentation beer in the present invention is preferably a sugar-deficient wort beer, a yeast-limited beer, a stopped fermentation beer or a cold contact fermentation beer. The limited fermentation beer in the present invention is preferably a cold contact fermentation beer.

[0037] In a much more preferred embodiment, the beer herein is a regular beer, a limited fermentation beer or a mixture of these two types of beer, preferably a regular beer, a cold contact fermentation beer or a mixture of these two types of beer. Most preferably, the beer herein is a regular beer as defined above, with an ethanol content of 1 to 15% by volume.

[0038] To obtain the optimal effect of improving foam stability, the beer of the present invention preferably has a low sugar content. The total sugar content of the beer of the present invention, defined as the sum of glucose, fructose, sucrose, maltose, and maltotriose, is preferably 0.05-5 g / 100 ml, more preferably 0.1-1 g / 100 ml, and even more preferably 0.15-0.5 g / 100 ml. This is because it has been found that the foam stabilizing effect of a reduced amount of AcHFA is enhanced in beers with a relatively low total sugar content.

[0039] For similar reasons, the beers of the invention preferably have a relatively high level of free amino nitrogen (FAN), which contributes to foam stability and therefore the beers of the invention preferably have an FAN content of 50-160 mg / l, preferably 90-140, more preferably 110-135 mg / l.

[0040] Isoalpha acids also have a positive effect on the foam stability of beer. Thus, the beer of the present invention preferably has a total of 2-55 mg / l, preferably 5-45 mg / l, more preferably 10-30 mg / l, more preferably 13-25 mg / l of isoalpha acids. Isoalpha acids are defined herein as the soft resin fraction of lupulin produced in female hop cones. Alpha acids include, for example, humulone, cohumulone, and adhumulone.

[0041] To obtain beer with reduced amounts of AcHFA as defined herein, three options are disclosed: removing AcHFA from beer during or after fermentation, avoiding AcHFA formation by using ATF-deficient yeast for fermentation, and removing AcHFA precursors from the mixture to be fermented. Thus, the present invention relates to a method for increasing the foam stability of beer, comprising the steps of fermenting a wort to obtain said beer, a) fermentation is accomplished using acetyltransferase (ATF) deficient yeast; and / or b) contacting the beer during or after fermentation with an adsorbent capable of adsorbing AcHFA, and / or c) the wort was subjected to a step of removing AcHFA precursors using an adsorbent capable of adsorbing AcHFA precursors; A method is disclosed.

[0042] ATF-deficient yeast In a preferred embodiment, fermentation is achieved using acetyltransferase (ATF)-deficient yeast (including any ortholog of ATF), which is preferably S. cerevisiae. Such yeast can be obtained by genetic modification of yeast (preferably S. cerevisiae), such as replacement or disruption (knockout) of ATF-encoding gene. In this specification, ATF-deficient yeast means that in a yeast type in which multiple copies of the ATF gene are present, preferably all copies of the ATF gene are knocked out.

[0043] There are two genes related to acetyltransferase activity (in S. Cerevisae), ATF-1 and ATF-2. The ATF-1 gene has gene ID number 854559 (NCBI database), and the ATF-2 gene has gene ID number 853088 (NCBI database). In other yeast types suitable for fermentation, common general knowledge can be used to identify the location of orthologous ATF genes. As mentioned above, in ATF-1-deficient yeast, preferably all copies of the ATF-1 gene are knocked out. In ATF-2-deficient yeast, preferably all copies of the ATF-2 gene are knocked out.

[0044] In one embodiment, the ATF-deficient yeast is an ATF-1-deficient yeast. ATF-1 deficiency results in a reduction of AcHFA formation of about 50-90%, preferably about 65-85%. In another embodiment, the ATF-deficient yeast is an ATF-2-deficient yeast. ATF-2 deficiency results in a reduction of AcHFA formation of about 1-40%, preferably about 10-30%. In a preferred embodiment, the ATF-deficient yeast is at least ATF-1-deficient, and preferably ATF-2-deficient. In a much more preferred embodiment, the ATF-deficient yeast is a yeast deficient in both ATF-1 and ATF-2. The use of yeast deficient in both ATF-1 and ATF-2 completely prevents the formation of AcHFA during fermentation.

[0045] The ATF-deficient yeast can be obtained by commonly known methods such as mutagenesis, preferably random mutagenesis, or genetic engineering.

[0046] As used herein, "mutagenesis" refers to a process in which at least one mutation is generated in the DNA of at least one yeast cell or its spore, thereby altering the genetic information of the yeast cell or spore. Thus, mutagenesis can produce the ATF-deficient yeast described above.

[0047] As used herein, the term "mutation" refers to any change in the DNA of a yeast cell or spore, including, but not limited to, point mutations, insertions or deletions of one or more nucleotides, substitutions of one or more nucleotides, frameshift mutations, and single- or double-stranded DNA breaks, such as chromosomal or subtelomeric breaks, and any combination thereof. Preferably, the mutation is located in the ATF gene to produce the ATF-deficient yeast described above.

[0048] Mutagenesis can be carried out using any method known in the art, including traditional random mutagenesis methods such as radiation and chemical treatment, as well as recombinant DNA techniques such as site-directed or targeted mutagenesis. Thus, in one embodiment, at least one yeast cell or spore of at least a first species of yeast is subjected to UV irradiation, X-ray irradiation, gamma irradiation, or treatment with a mutagen, or genetic engineering.

[0049] "Genetic engineering" is well known in the art and refers to the use of biotechnology to introduce changes in the DNA of yeast cells or spores to alter the genome of the yeast cells or spores.

[0050] "Random mutagenesis" refers to a mutagenesis technique in which the exact site of mutation is not predictable and can occur anywhere in the chromosome of yeast cells or spores. Generally, these methods involve the use of chemicals or radiation to induce at least one mutation. Random mutagenesis can further be achieved using error-prone PCR, where the PCR is performed under conditions in which the DNA polymerase has low copying accuracy and the mutation rate in the PCR product is relatively high. Site-specific mutagenesis can be achieved using oligonucleotide-directed mutagenesis to generate site-specific mutations in the DNA sequence of interest. Targeted mutagenesis refers to a mutagenesis method in which a specific or targeted gene is altered in vivo by programmable RNA-guided nucleases such as TALEN, CRISPR-Cas, zinc finger nucleases or meganuclease technology, resulting in a genetic structural change directed to a specific site.

[0051] In a preferred embodiment, mutagenesis in the method of the invention is carried out by subjecting at least one yeast cell or spore to radiation, such as UV irradiation, X-ray irradiation, gamma irradiation, or treatment with a mutagen, preferably a chemical such as NTG (N-methyl-N'-nitro-N-nitrosoguanidine) or EMS (ethyl methanesulfonate).

[0052] Adsorption of AcHFA or AcHFA precursors In a further preferred embodiment, the fermented beer is contacted with an adsorbent capable of adsorbing AcHFA. Additionally, AcHFA precursors can be removed from the wort prior to fermentation by contacting the wort with an adsorbent capable of adsorbing AcHFA precursors.

[0053] It has been found that the same adsorbent can be used for removing AcHFA precursors from wort and for removing AcHFA from beer during or after fermentation. That is, AcHFA precursors and AcHFA itself are substantially adsorbed on the same adsorbent. Suitable adsorbents include activated carbon, hydrophobic adsorbents, hydrophilic adsorbents, and zeolites. Preferably, these adsorbents are applied during or after fermentation (preferably after fermentation) to adsorb AcHFA from beer.

[0054] To improve the foam stability of unhopped beer, the beer may be contacted with any adsorbent suitable for adsorbing AcHFA (or its precursors). Thus, in a preferred embodiment, the beer is unhopped. Optionally, hops may be added to the beer later to achieve further foam stability improvement and further (flavor) benefits. Hopped beer contains iso-alpha acids that have an additional stabilizing effect on the beer foam.

[0055] To improve the foam stability of hopped beer, the beer is preferably contacted with an adsorbent that does not substantially adsorb isoalpha acids. Isoalpha acids are obtained from hops by wort boiling and are known to have a foam stabilizing effect. Those skilled in the art recognize that any adsorbent can be used in hopped beer as long as the adsorbent has a final improving effect on foam stability. This may depend on the amount of hops used and / or the amount of AcHFA formed during fermentation. In a preferred embodiment, the hopped beer can be further hopped after the AcHFA removal step is completed.

[0056] Activated carbon is well known in the art and can be used as an adsorbent to remove AcHFA and obtain the AcHFA-reduced beer according to the present invention. However, a drawback of activated carbon is that it is not very efficient at removing AcHFA. In addition, activated carbon significantly adsorbs iso-alpha acids, which is a drawback in the processing of hopped beer. A further drawback is that activated carbon decolorizes the beer, resulting in a beverage that is too light in color and unappealing to consumers.

[0057] Hydrophobic adsorbents include hydrophobic polymer adsorbents, preferably polymer adsorbents containing aromatic and / or acyl groups. Based on common general knowledge and the methods described herein, a person skilled in the art can easily determine which adsorbents are suitable for reducing AcHFA (or its precursors) from beer (or wort). The advantage of hydrophobic polymer adsorbents is that they are very efficient in removing AcHFA (or AcHFA precursors). In addition, the color of the beer is preserved, and these adsorbents adsorb relatively little or even no isoalpha acids. Therefore, hydrophobic polymer adsorbents are preferred, especially polystyrene / divinylbenzene (PS / DVB), e.g. PLRP-S (sold by Agilent).

[0058] The hydrophilic adsorbent is preferably a hydrophilic polymer adsorbent. A much more preferred hydrophilic adsorbent is a mixed cellulose ester adsorbent, such as MCE (MF-Millipore™ Membrane Filter, Merck). It is the most efficient adsorbent tested, leaving the color of the beer unchanged and does not adsorb iso-alpha acids. Moreover, this adsorbent can be regenerated, leading to lower production costs and less waste, especially in the beer industry.

[0059] Further preferred adsorbents are Supelclean™ adsorbents, such as, for example, preferably Supelclean LC C18, Supelclean LC C8, Supelclean LC Ph, Supelclean LC CN, or Supelclean LC SCX, more preferably Supelclean LC C18, Supelclean LC C8, or Supelclean LC Ph, most preferably Supelclean LC C18, Supelclean LC C8, etc. These adsorbents are preferred for the same reasons as those mentioned above for MCE and PLRP-S.

[0060] The zeolite herein is preferably a hydrophobic zeolite, which is SiO 2 and Al 2 O 3 at a molar ratio of at least 15 (SiO 2 :Al 2 O 3 ) As used herein, the term "molecular sieve" refers to a microporous material having pores with diameters of 2 nm or less. The term "silicate-based" means that the material contains at least 67% by weight of silicate. Thus, a "zeolite" is a microporous aluminosilicate. The zeolite adsorbent herein can be a natural zeolite or a synthetic zeolite.

[0061] SiO 2 Contains Al 2 O 3 Hydrophobic silicate-based molecular sieves that do not contain SiO 2 and Al 2 O 3 in a molar ratio of at least 15 (in which case the zeolite is a microporous silicate rather than a microporous aluminosilicate).

[0062] According to a preferred embodiment, the adsorbent is a hydrophobic zeolite. The hydrophobic zeolite employed in the present process is preferably SiO 2 / Al 2 O 3 is at least 40, more preferably at least 100, even more preferably at least 200, and most preferably at least 250.

[0063] The average pore diameter of the hydrophobic zeolite is preferably in the range of 0.2 to 1.2 nanometers, more preferably 0.3 to 1.0 nanometers, even more preferably 0.4 to 0.8 nanometers, and most preferably 0.45 to 0.70 nanometers. The pore diameter of the hydrophobic zeolite can be determined by analyzing the nitrogen adsorption isotherm at 77K by the t-plot-De Boer method.

[0064] The surface area of ​​the hydrophobic zeolite is preferably at least 100 m 2 / g, more preferably 150 to 2000m 2 / g, most preferably 200 to 1000m 2 The surface area of ​​the hydrophobic molecular sieve can be determined by the BET method.

[0065] The hydrophobic zeolite preferably has a mass weighted average particle size in the range of 1 to 2000 micrometers, more preferably in the range of 10 to 800 micrometers, and most preferably in the range of 100 to 300 micrometers. The particle size distribution of the hydrophobic molecular sieve particles can be determined using a set of sieves of different mesh sizes.

[0066] The hydrophobic zeolite is preferably selected from ZMS-5 zeolite, Y-type zeolite, zeolite beta, silicalite, all-silica ferrierite, mordenite, and combinations thereof. More preferably, the hydrophobic zeolite is selected from ZMS-5 zeolite, Y-type zeolite, zeolite beta, and combinations thereof. Most preferably, the hydrophobic zeolite is ZMS-5 zeolite.

[0067] Adsorbents for use as disclosed herein can be applied as known in the art.

[0068] To remove AcHFAs during fermentation, the fermentation mixture can be contacted with an adsorbent, for example by passing the mixture through a packed column of adsorbent, or by adding the adsorbent as a particulate material to the fermentation mixture and subsequently removing the adsorbent by filtration, cycloning, or another suitable technique.

[0069] For the removal of AcHFAs after fermentation, the beer obtained from fermentation can be contacted with an adsorbent, for example by passing the beer through a column packed with the adsorbent, or by adding the adsorbent as a particulate material to the beer, followed by removal of the adsorbent by filtration, cycloning or another suitable technique. In a preferred embodiment, the beer obtained from fermentation is introduced into a hold-up tank, where it is contacted with an adsorbent for adsorption of AcHFAs, for example for 20 minutes to 24 hours, preferably 0.5 to 5 hours. The AcHFAs can then be removed by filtration. Alternatively, the beer can be continuously filtered through an adsorbent-loaded filter.

[0070] The AcHFA precursors can be removed by contacting the wort with an adsorbent prior to fermentation, for example by passing the wort through a packed column of adsorbent, or by adding the adsorbent to the wort as a particulate material and subsequently removing the adsorbent by filtration, cycloning, or another suitable technique. Preferably, the wort is contacted as a sweet wort prior to pitching.

[0071] Those skilled in the art are aware of numerous techniques for adsorbing undesirable components from liquid mixtures by adsorption, any of which may be applied to obtain beer with reduced amounts of AcHFA as defined herein. Suitable techniques are described, for example, in C. Judson King, Separation Processes (2nd Edition), McGraw-Hill, Inc., 1980.

[0072] The wort, fermentation mixture or beer is contacted with the adsorbent for a time sufficient to adsorb AcHFA or its precursors, which can be readily determined by one of ordinary skill in the art based on common general knowledge and the methods disclosed herein. Preferably, the contact time is between 5 minutes and 48 hours, more preferably between 0.5 and 24 hours, more preferably between 1 and 20 hours.

[0073] The amount of adsorbent used can also be easily determined by a person skilled in the art based on common general knowledge and the methods disclosed herein. Preferably, the adsorbent is used in a dosage of 0.001 to 10 g / l, more preferably 0.01 to 5 g / l, more preferably 0.05 to 2.5 g / l.

[0074] Depending on the type and nature of the beer, the type and amount of adsorbent, and the contact time, the amount of AcHFA can be substantially reduced, as can be readily ascertained by one skilled in the art. The amount of AcHFA can be reduced by at least 50%, preferably at least 75%, more preferably at least 80%. In some embodiments, the amount of AcHFA can be reduced by up to 90%, or even up to 95% or more. In a preferred embodiment in which AcHFA is removed from the beer by adsorption, the amount of AcHFA is reduced to less than 60% of the initial amount, more preferably less than 20% of the initial amount, and most preferably less than 5% of the initial amount.

[0075] The effect of AcHFA reduction is that foam stability is increased by at least 10 seconds, preferably at least 15 seconds, and more preferably at least 20 seconds. Foam stability can be increased by up to 40 seconds, or even up to 50 seconds or more.

[0076] In a much more preferred embodiment, the present invention relates to a method for increasing the foam stability of beer, comprising the step of contacting beer obtained by fermentation with an adsorbent capable of adsorbing AcHFA, the adsorbent and the manner of contacting the beer with the adsorbent being as defined above, resulting in a beer with said increased foam stability.

[0077] Although features are described herein as part of the same or separate embodiments for clarity and conciseness of description, it is recognized that the scope of the invention can include embodiments having combinations of all or part of the described features.

[0078] The invention will now be further illustrated by the following non-limiting examples.

[0079] method AcHFA analysis The AcHFA content in beer can be analyzed by LC-MS. The LC-MS system consisted of a Waters TQ-S mass spectrometer and a Waters Acquity UPLC system. The mobile phase was: A: Milli-Q water + 0.1% (v / v) formic acid B: Acetonitrile + 0.1% (v / v) formic acid It consisted of the following:

[0080] The analytical column was a 15 cm x 2.1 mm ID UPLC BEH C18 (1.7 μm) column. AcHFA was separated from other matrix components with the following gradient: T 0 min: 95% A, 5% B T 13 minutes: 70% A, 30% B T 17 minutes: 5% A, 95% B T 25 minutes: 95% A, 5% B

[0081] The flow rate was set at 0.25 ml / min and the column was thermostatted at 50° C. The UPLC system was coupled to the MS via an electrospray interface (ESI) operated in negative ion mode. MRM data was recorded for the following transition: m / z 355>295. The cone voltage was set at 40 volts. The collision energy was set at 15 eV. AcHFA was detected as the compound represented by structure 3. The amount of AcHFA was expressed in arbitrary units (au), which are derived from the peak surface of the chromatogram and provide quantitative results.

[0082] Determination of sugar content in beer The sugar content was measured by ultra-performance liquid chromatography (UPLC). UPLC can be suitably carried out at a temperature of 65° C. A suitable choice of eluent is a mixture of acetonitrile / water, for example in a volume ratio of 75 / 25. The detector used is typically a refractive index (RI) detector. The sugar content of the sample was determined by comparing the UPLC curve of the sample with a calibration curve of standard samples of known sugar concentration.

[0083] Samples for UPLC were prepared as follows: Beer or wort samples were diluted 5-fold by addition of a mixture of acetonitrile / water (50 / 50-equal parts by volume). 2 If present, it was removed (e.g., by shaking or stirring the sample) prior to dilution. After dilution, the sample was filtered to obtain a clear solution. The filtered sample was injected into the UPLC at 65° C. using the eluent described above.

[0084] Determination of free amino nitrogen (FAN) The amount of free amino nitrogen (amino acids, small peptides and ammonia, etc.) was measured according to the Nitrogen by O-phthaldialdehyde Assay (NOPA) method. The NOPA method was carried out using a photometric analyzer (e.g., Gallery™ Plus Photometric Analyzer). According to the NOPA method, the test sample is subjected to treatment with ortho-phthaldialdehyde (OPA) and N-acetylcysteine ​​(NAC). This treatment results in the derivatization of the primary amino groups present in the test sample under the formation of isoindole. The content of isoindole is subsequently determined at a wavelength of 340 nm using a photometric analyzer. The free amino nitrogen (expressed in mg FAN / l) can then be calculated based on the measured value of the isoindole content. If necessary, the beer or wort sample is first centrifuged before analysis to clarify the sample and / or to reduce CO (e.g., by stirring or shaking the sample). 2 Subject to removal step.

[0085] Determination of ethanol in beer Ethanol content was measured using a photometric analyzer (e.g., Gallery™ Plus Photometric Analyzer). Test samples are subjected to an enzymatic method in which ethanol present in the sample is converted to acetaldehyde by alcohol dehydrogenase (ADH). The acetaldehyde content is then determined using a photometric analyzer at a wavelength of 340 nm. The ethanol content can be calculated based on the acetaldehyde content. If necessary, the beer or wort samples can be first centrifuged to clarify the sample and / or CO2 (e.g., by stirring or shaking) prior to analysis. 2 Subject to removal step.

[0086] Determination of iso-alpha acids in beer EBC 9.47 2010: Quantitative determination of iso-alpha-acids using Iso-α-acids and reduced iso-α-acids in beer by HPLC (Rho, Tetra, Hexa).

[0087] Determination of foam stability Foam stability is determined according to the criteria set by the NIBEM Institute, EBC 9.42.1.

[0088] Deaeration and recarbonation All experiments involving AcHFA adsorption from beer were performed with degassed regular beer. The beer was recarbonated before foam stability measurements.

[0089] Degassed beer was obtained as follows.

[0090] Approximately 200 ml of bottled beer (4° C.) was carefully transferred (to prevent foaming) into a clean 500 ml laboratory bottle. The bottle containing the sample was placed in a 20° C. water bath and shaken gently (40 rpm) for 40 minutes.

[0091] The shaking intensity was then increased to 120 rpm and shaking was continued for 30 minutes to obtain degassed beer. During the entire degassing process, the headspace of the bottle was continuously flushed with nitrogen gas to prevent oxidation of the beer.

[0092] Recarbonation was accomplished on a laboratory scale as follows. A stainless steel Millipore vessel (model Millipore catalog number xx6700p05) was prepared to serve as a regular beer keg. Prior to the addition of beer, the keg was cleaned and filled with CO 2 Approximately 1-2 liters of beer sample was transferred to a keg and flushed with 2.5 bar CO while shaking the sample at 100 rpm for 30 minutes. 2 The entire procedure was carried out at 20°C.

[0093] CO 2 Using pressure, the beer was dispensed using a spear positioned approximately 0.7 cm from the bottom of the container. Thus, the Nibem foam dispenser device can be used with a similar (CO 2Foams were prepared by connecting the flask to a pressurized (pressurized) barrel. Foam stability was determined according to the Nibem method described above. EXAMPLES

[0094] Removal of AcHFA from beer results in foam stabilization The degassed beer was incubated for 16 h with 0.1 g / l of a commercial methylcellulose ester adsorbent (MCE, MF-Millipore™ membrane filter, Merck) at room temperature. The beer was then filtered through a 0.2 μm filter and recarbonated.

[0095] The results show that adsorption of AcHFA onto MCE followed by removal of AcHFA from the beer results in a beer with improved foam stability. Blank beer was subjected to the same procedure except for the adsorption step. After treatment, AcHFA in the treated beer was reduced to 6.3% of the amount in the regular beer. The blank beer had a (NIBEM) foam stability of 253 seconds while the MCE treated beer had a foam stability of 296 seconds. Thus, removal of over 90% of AcHFA resulted in a 17% increase in foam stability.

[0096] The AcHFA removal process has been shown to result in efficient removal of AcHFA in both hopped and unhopped beers (data not shown). Furthermore, it was found that MCE does not significantly adsorb iso-alpha acids and the color of the beer is not reduced. This is beneficial since the presence of iso-alpha acids has an additional stabilizing effect on the beer foam. Therefore, MCE is by far the preferred adsorbent. EXAMPLES

[0097] AcHFA is a negative factor for foaming A degassed regular beer was obtained as described in Example 1. The beer in this case was a full malt beer without hops. The degassed beer was incubated with 0.2 g / l PLRP-S (Agilent) at room temperature for 16 hours to ensure complete removal of AcHFA and to obtain an AcHFA-free unhopped beer.

[0098] The sorbent was filtered and isolated, washed with acetonitrile to obtain crude AcHFA. The crude AcHFA was fractionated on a reverse-phase HPLC column (C18) using a water to acetonitrile gradient. The resulting fractions were tested for their effect on foam stability by addition to beer, and the foam negative fractions were collected, analyzed by LC-MS, and characterized as AcHFA by NMR as defined in the specification. Thus, isolation and characterization of pure AcHFA was performed.

[0099] An additional amount of pure AcHFA was added to deaerated hopped and deaerated unhopped beers with regular amounts of AcHFA obtained from normal fermentation. After gentle homogenization and recarbonation as described in Example 1, foam stability was determined as described. The amount of AcHFA added is shown in Table 1 and depicted in Figures 2a and 2b.

[0100] [Table 1]

[0101] The results show that by increasing the amount of AcHFA in regular beer, the foam stability of the beer rapidly decreases. Therefore, it is concluded that AcHFA is a negative factor for foam, and beer with reduced AcHFA content has increased foam stability. EXAMPLES

[0102] Beer with reduced AcHFA content has increased foam stability In a separate experiment, AcHFA-free hopped and AcHFA-free unhopped beers were obtained by incubation with 0.2 g / l PLRP-S (Agilent) at room temperature for 16 hours to achieve complete removal of AcHFA. Prior to the addition of AcHFA to the hopped beer, the loss of iso-alpha acids during the adsorption step was compensated for by the addition of 20 mg / l iso-alpha acids ("IAA", B-Hop, Hopsteiner). Both AcHFA beers were subsequently degassed.

[0103] Varying amounts of AcHFA were added to AcHFA-free beer. After gentle homogenization and recarbonation as described above, foam stability was determined. The results are shown in Table 2 and Figures 3a and 3b.

[0104] [Table 2]

[0105] The results show that by reducing the amount of AcHFA in the beer below the levels in regular beer, foam stability increases in an AcHFA concentration-dependent manner, with AcHFA-free beer having the highest foam stability. EXAMPLES

[0106] Reducing AcHFA in beer Regular (hop-free) beer with conventional levels of AcHFA was obtained. The beer was degassed as described and 30 ml aliquots (10 column volumes) were contacted with the same amount of various Supelclean™ sorbents (Sigma Aldrich) in 3 ml SPE columns followed by recarbonation. The results are shown in Table 3 and Figure 4.

[0107] [Table 3]

[0108] The results show that various hydrophobic and hydrophilic adsorbents can reduce the amount of AcHFA in beer. Preferred adsorbents are LC C18, LC C8 and LcPh. In a preferred embodiment, AcHFA is reduced to less than 60% of the initial amount, more preferably less than 20% of the initial amount, even more preferably less than 5% of the initial amount. EXAMPLES

[0109] Reduction of AcHFA in beer using PLRP-S and activated carbon Hop-free deaerated beer was contacted with PLRP-S (0.1 mg / l) and 1.0 mg / l activated carbon (Aktivkohle, art. 2186 Merck) followed by recarbonation. The results are shown in Figure 5.

[0110] The results show that PLRP-S is more efficient in removing AcHFAs from beer. Furthermore, it was observed that activated carbon decolorizes the beer, which is considered a drawback. PLRP-S leaves the color of the beer unchanged. Furthermore, PLRP-S does not significantly adsorb iso-alpha acids. Therefore, PLRP-S (and MCE, see Example 1) are preferred adsorbents over activated carbon. EXAMPLES

[0111] Removal of AcHFA precursors from sweet wort to obtain beer with reduced AcHFA content compared to conventional beer In a conventional pilot plant beer brewing process, sweet wort was treated with PLRP-S by coating a PVPP filter placed between the lauter tun and the copper wort vessel with 750 g of PLRP-S and treating 15 hl of sweet wort through said filter prior to entering the copper wort vessel (Figure 6). The beer was then brewed conventionally (hop addition, boiling, whirlpooling, fermentation, and post-processing and bottling).

[0112] A comparison beer was brewed from the same wort using the same process conditions but without filtering the sweet wort through the PLRP-S loaded filter.

[0113] Analysis of the amount of AcHFA precursors (dihydroxy fatty acids) was performed on samples of hopped wort (pitched wort) using the HPLC conditions described for AcHFA. The amount of AcHFA was determined for the final beer. The results are shown in Table 4.

[0114] [Table 4]

[0115] The results show that the removal of AcHFA precursors by adsorptive action can prevent the formation of AcHFA in the final beer, which results in increased foam stability of the resulting beer.

[0116] The results further demonstrate that the adsorption process can be applied on an industrially relevant scale. EXAMPLES

[0117] To obtain beer with reduced AcHFA content by modifying yeast In this example, a deletion library of S. pastorianus wild-type (WT) yeast with different combinations of deletion alleles of ATF1 and ATF2 was constructed using CRISPR-Cas9 technology. S. pastorianus WT contains five copies of ATF1 and four copies of ATF2 from both the S. cerevisiae and S. eubayanus genomes. Using the functional CRISPR-Cas9 system in S. pastorianus, all copies of a gene are targeted in a single transformation. Finally, nine ATF1 / 2 genes were deleted from four different chromosomes in just two transformations. The ATF-1-deficient yeast is referred to as "strain a". The ATF-2-deficient yeast is referred to as "strain b". The yeast deficient in both ATF-1 and ATF-2 is referred to as "strain c". The effects of the various knockouts were investigated by growing the strains under brewing-relevant conditions. The strains exhibited similar growth rates, sugar consumption profiles, and ethanol production during standard brewing fermentation in 15° Plateau wort.

[0118] Obtaining mutants from wild-type yeast The mutant strains were obtained according to the method described in “CRISPR-Cas9 mediated gene deletions in lager yeast Saccharomyces pastorianus”, Arthur R. Gorter de Vries et al., Microb Cell Fact (2017) 16:222. The designation of the mutations is shown in Figure 7a.

[0119] Characterization of WT with ATF deficiency under brewing conditions To check the fermentation performance, the strains were inoculated into 15° plateau wort that had been autoclaved and filter sterilized and supplemented with zinc to meet the yeast growth requirements. Three bottles per strain were inoculated with the same cell concentration of about 5 million cells. All bottles were sampled at the beginning of the experiment and after a few days of fermentation for analysis by HPLC. However, one bottle out of each three bottles was also sampled during fermentation to monitor sugar consumption and glycerol, ethanol, and biomass production and compare with the fermentation with the WT. After fermentation, the supernatant of each bottle was analyzed by HPLC for sugar, glycerol, and ethanol. It was observed that the mutant strains behaved virtually identically to the wild-type yeast when applied in fermentation. The sugar consumption profiles over time for glucose, maltose, maltotriose, and fructose show that virtually identical consumption profiles are used. Also, the glycerol and ethanol production monitored during fermentation was virtually identical. Also, the cell growth rates (measured by optical density) were virtually identical. An exemplary diagram showing the ethanol production of strains a, b and c is incorporated as Figure 7b. For all test parameters, the mutant strains showed substantially identical characteristics and substantially identical time courses. Therefore, the mutant strains can be applied to existing production processes to reduce the amount of AcHFA in the final beer.

[0120] Thus, ATF-deficient yeast types were obtained that could be applied under the same conditions as the WT yeast. The ATF-1-deficient yeast (strain a), the ATF-2-deficient yeast (strain b) and the yeast deficient in both ATF-1 and ATF-2 (strain c) were used in standard fermentation of wort to evaluate their activity in the standard beer brewing process. The different yeasts inoculated with 5 mio cells / ml were fermented in 250 ml percolation vessels in 15-16 P wort with 150 ml of full malt hops at 200 rpm, 13 °C and shaking under microaerobic conditions for 7 days. The obtained beer was compared with the beer obtained with the unmodified WT yeast. The results are shown in Table 5.

[0121] [Table 5]

[0122] The results show that the use of ATF-1-deficient yeast reduces AcHFA to 15% of the amount of AcHFA observed using wild-type yeast. The use of ATF-2-deficient yeast reduces AcHFA to 77% of the amount of AcHFA observed using wild-type yeast. By using yeast that is deficient in both ATF-1 and ATF-2 (preferably all copies), AcHFA formation can be completely suppressed. EXAMPLES

[0123] Removal of AcHFA from regular beer on an industrially relevant scale to obtain beer with reduced AcHFA content Beer was brewed in a pilot plant using a conventional process and conventional yeast. The brewing process resulted in lager beer. Lager beer with added iso-alpha acids ("IAA") (20 mg / l B-Hop, Hopsteiner) and beer without added iso-alpha acids were obtained.

[0124] Lager beer was filtered, stabilized with PVPP, and stored in a hold-up tank in contact with 0.1 g / l PLRP-S (Agilent) for 1 or 2 h. The beer was subsequently filtered to remove PLRP-S and adsorbed AcHFA (Figure 8a). Alternatively, the beer resulting from fermentation could be filtered through a PLRP-S coated filter to remove AcHFA (Figure 8b; data for the resulting beer is not shown but is comparable to that for the beer obtained by the process displayed in Figure 8a).

[0125] The amount of AcHFA was analyzed and compared to that of beer without the filtration step. Additionally, the foam stability of the resulting beers was compared.

[0126] The results show that the amount of AcHFA can be significantly reduced by treating the beer resulting from fermentation with PLRP-S for 1 or 2 hours. This had a significant positive effect on the foam stability of beer with and without added iso-alpha acids. Therefore, adsorbents such as PLRP-S can be used to remove AcHFA and increase the foam stability of beer.

Claims

1. 1. A beer containing less than 0.5 mg / l AcHFA, AcHFA is a C12-C22 fatty acid comprising a carboxylic acid group and a C11-C21 linear alkyl group, said alkyl group being optionally partially unsaturated and substituted with at least one hydroxy group and at least one acetate group; AcHFA is defined by Structure 1, 【Chemistry 1】 In Structure 1, n=an integer from 4 to 9; 【Chemistry 2】 , each of A, B, C and / or D may be the same or different; a) 【Chemistry 3】 is a single bond, in which case: One of A and B is H, OH or OAc, and the other of A and B is H; one of C and D is H, OH or OAc and the other of C and D is H; or b) 【Chemistry 4】 is a double bond, in which case, One of A and B is H, and the other of A and B does not exist; one of C and D is H, and the other of C and D does not exist; beer, with the proviso that in structure 1, at least one of every A, B, C, D is OH, and at least one of every A, B, C, D is OAc.

2. In the beer according to claim 1, AcHFA is defined by structure 2, 【Chemistry 5】 In Structure 2, n=1, 2, or 3 and m=1 or 2; One of A and B is OH and the other of A and B is OAc; Beer.

3. 3. The beer of claim 2, wherein in Structure 2, n=2 or 3 and / or m=2.

4. 4. The beer of claim 3, wherein in structure 2, n=3 and m=2.

5. In the beer according to any one of claims 1 to 4, Beer containing 0 to 15% by volume of ethanol.

6. In the beer according to any one of claims 1 to 5, Beer containing less than 0.25 mg / l AcHFA.

7. In the beer according to any one of claims 1 to 6, Beer containing 5-55 mg / l total iso-alpha acids.

8. In the beer according to any one of claims 1 to 7, Beer having a total sugar content, defined as the sum of glucose, fructose, sucrose, maltose, and maltotriose, of 0.05 to 5 g / 100 ml.

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