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    • 专利摘要:
    The present invention relates to a beer with reduced amounts of AcHFA. Beer with reduced AcHFA amounts has been found to have improved foam stability. The invention provides beer with reduced AcHFA amounts, as well as methods for removing AcHFA from beer during or after fermentation, or for removing a precursor for AcHFA from wort.
    公开号:BE1026596B1
    申请号:E20195600
    申请日:2019-09-10
    公开日:2020-04-06
    发明作者:Augustinus Cornelius Aldegonde Petrus Albert Bekkers；Eric Richard Brouwer
    申请人:Heineken Supply Chain Bv；
    IPC主号:
    专利说明:

    Foam stability
    The invention relates to a beer with improved foam stability, as well as to methods for improving the foam stability of beer.
    Background
    Beer is one of the most popular alcoholic drinks worldwide. It is prepared by fermentation of a sugary aqueous matrix derived from grains, using yeast that converts the sugars into ethanol ("alcohol"). The beer production process is well known, and those skilled in the art are able to obtain beer based on common knowledge (see, for example, The Brewer's Handbook (second edition) by Ted Goldammer (2008, Apex Publishers) and the information disclosed herein.
    Beer is usually made from grain such as barley, although other grain types including but not limited to wheat or sorghum can also be used. Beer is usually produced by a process that includes the following basic steps: mashing a mixture of grain and water to produce a mash; separating the mash into wort and brewing; cooking the wort to produce a boiled wort; fermenting the cooked wort with live yeast (such as Saccharomyces pastorianus or Saccharomyces cerevisiae) to produce a fermented wort; subjecting the fermented wort to one or more further process steps (e.g. maturation and filtration) to produce beer; and packaging the beer in a sealed container, e.g. a bottle, can or barrel.
    In an exemplary process to produce a barley malt beer, the barley is malted, which means that it is germinated and then dried ('eesten') to produce malt. This process is important for the formation of flavor and color compounds, and formation
    BE2019 / 5600 of enzymes that are important for further flavor development and starch breakdown. The malt is then ground and suspended in water ('mashing'). The maize is heated to facilitate starch breakdown. Subsequent filtration results in wort, which is a more or less clarified aqueous solution of fermentable sugars, which also contains various flavors and aromas and many other compounds. Both desirable and undesirable flavor compounds are present in wort.
    The wort is boiled to sterilize it, to precipitate proteins and to concentrate it. Hops are optionally added to add bitterness and flavor. This mixture, after removal of the precipitate, is subjected to fermentation. Fermentation results in the conversion of fermentable sugars into ethanol and carbon dioxide, and also results in the formation of various new flavor compounds. At the same time, the yeast used for the fermentation accomplishes many other chemical conversions. After fermentation, the beer can be filtered and / or stored to optimize appearance and taste.
    An important aspect of beer is the beer collar. The collar is a layer of foam on top of the beer. This foam arises from carbon dioxide that is present in the beer through the fermentation and / or post-fermentation addition, but which is essentially dissolved in the beer due to the high pressure in the beer container (e.g. a can or bottle). Releasing the beer from the container into, for example, a glass causes the formation of carbon dioxide bubbles, which rise through the beer liquid to the top of the glass to form a foam.
    A distinctive feature of beer foam is its stability. Unlike other foaming yeast fermented drinks (eg champagne), beer foam is stable. This is caused by (among other things) proteins and isomerized hop acids in beer, which position themselves at the interface of the bubble and the liquid during the formation and rise of the carbon dioxide bubble through the beer. At the
    BE2019 / 5600 reaching the top of the liquid, these components stabilize the beer foam bubbles. This keeps the foam layer intact for a long period of time. An intact and stable foam layer is considered an important aspect of a pleasant beer.
    The foam stabilization is, however, of limited duration. Within a few minutes after pouring, the foam layer becomes thinner, and eventually there is no foam at all. It is possible that this process occurs faster than the user wants to drink the beer. This means that the beer is less enjoyed over time due to a disappearing foam layer. For slow drinkers, the foam may have already disappeared before the beer is drunk.
    The present invention provides beers with improved foam stability, and methods to improve the foam stability of beer.
    Summary
    The present invention relates to a beer with reduced amounts of AcHFA. Beer with reduced AcHFA amounts has been found to have improved foam stability. The invention provides beer with reduced AcHFA amounts, as well as methods for removing AcHFA from beer during or after fermentation, or removing a precursor for AcHFA from wort.
    Figures
    Figure 1: Comparison in foam stability before and after an adsorption process with a methyl cellulose ester adsorbent.
    Figure 2: The effect of AcHFA addition on foam stability of hopped (2a) and unhopped (2b) beer.
    Figure 3: The effect of AcHFA removal from hopped (3a) and unhopped (3b) beer.
    Figure 4: AcHFA removal from beer by a variety of adsorbents.
    BE2019 / 5600
    Figure 5: The activity of two adsorbents in removing AcHFA from beer.
    Figure 6: Installation of an adsorbent filter in an industrial-scale beer brewing process.
    Figure 7a: ATF-1 mutations in yeast.
    Figure 7b: ethanol production of ATF-1 deficient yeast (strain a), ATF-2 deficient yeast (strain b), and yeast deficient in both ATF-1 and ATF-2 (strain c), compared to the unmodified yeast (WT).
    Figure 8a and b: exemplary setups to remove AcHFA from beer on a factory scale.
    Figure 9: Increased foam stability in beer produced using a factory scale.
    Detailed description
    The invention provides a beer comprising less than 2 mg / l AcHFA, wherein AcHFA is a C12 - C22 fatty acid, comprising a carboxylic acid group and a C1 - C21 linear alkyl group, which alkyl group may be partially unsaturated and which alkyl group is substituted with at least one hydroxyl group and at least one acetate group. Preferably, the beer comprises less than 1.5 mg / l AcHFA, 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. Thus, the invention relates to beer comprising at least less than 10 mg / 1, such as less than 9 mg / 1, less than 8 mg / 1, less than 7 mg / 1, less than 6 mg / 1, less than 5 mg / 1, less than 4 mg / 1 AcHFA, or less than 3 mg / 1 /.
    AcHFA has now been shown to form during regular fermentation of wort by yeast, due to the action of the enzyme acetyl transferase (ATF). It has been found to be normally present in beer obtained by fermentation, at a level of at least 2 mg / 1, sometimes at least 3 mg / 1, or even at least 4 mg / 1, or at least 5 mg / 1, or at least
    BE2019 / 5600 mg / 1, or at least 7 mg / 1, or at least 8 mg / 1, or at least 9 mg / 1, or at least 10 mg / 1. Thus, AcHFA in beer obtained by fermentation can be present in amounts higher than 1 mg / l, in any case higher than 0.5 mg / l, and certainly higher than 0.25 mg / l. The inventors found that AcHFA is a foam-negative factor: it has a negative effect on foam stability. By adjusting the fermentation process to decrease the amount of AcHFA in the final beer, foam stability can be increased by at least 10%, up to 40%.
    AcHFA ("acetylated hydroxy fatty acid" [acetylated hydroxy fatty acid]) is a C12 -C22 fatty acid, comprising a carboxylic acid group and a C1-C21 linear alkyl group, which alkyl group may be partially unsaturated and which alkyl group is substituted with at least one hydroxyl group and at least one acetate group. An acetate group (H3CCO2 ~) is abbreviated (as is common in the art) as ~ OAc.
    AcHFA can be defined as structure 1
    where n = is an integer in the range of 4-9, and where each, A, B, C, and / or D may be the same or different, and where either
    a) 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) is a double bond, in which case:
    one of A and B is H, while the other of A and B is not present (meaning that the other of A and B is nothing), and one of C and D is H,
    BE2019 / 5600 while the other of C and D is not present (meaning that the other of A and B is nothing);
    provided that in structure 1 at least one of all A, B, C, D is OH and at least one of all A, B, C, D is OAc.
    As is well known, the double bond can have a cis or trans configuration, but the configuration is preferably cis. Furthermore, as is customary for organic acids, the acid group can be in neutral form (as shown; -CO2H), but can also be in ionic form (~ CO2 '), or in salt form ((~ CO2) X M, where M any metal ion can be, and preferably a metal ion available in beer, such as, for example, an ion of Na, K, Ca, Mg, Fe, Cu, Zn or Mn, and where x = 1 if M is monovalent (Na or K), and where x can be 1, 2 or 3 for higher valency ions Carbon atoms bearing OH or OAc can independently have a 7 or S configuration, but adjacent carbon atoms carrying an OH and an OAc group preferably both have a 7 configuration, or both have an S configuration (RR and SS) Alternatively, one carbon of adjacent carbon atoms carrying an OH and OAc group has an S configuration, and the other carbon of the two adjacent carbon atoms have a 7 configuration (RS or SR).
    Preferably, AcHFA includes one hydroxyl group and one acetate group located on adjacent carbon atoms, among the optional multiple hydroxyl and / or acetate groups. Furthermore, AcHFA is preferably a C16 - C20 fatty acid (n = 6-8 in structure J), most preferably a C18 fatty acid (n = 7 in structure Γ). It is very preferred if AcHFA comprises one or two double bonds, preferably one double bond. Preferably, double bonds are located on the 6th, 9th, 12th or 15th carbon atom, counting from the carboxylic acid group. Most preferably, a double bond is located on the 9th carbon atom.
    In highly preferred embodiments, AcHFA is represented by structure 2:
    BE2019 / 5600
    a
    HO
    CH 3 where 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.
    Also for structure 2, the double bond can have a cis or trans configuration (depicted by the line format ΜΛΜΜΜΛ ., Which indicates that the orientation of a single carbon-carbon bond extending from a double carbon-carbon bond can be in any direction), but preferably the configuration is cis. The acid group in structure 2 may be in neutral form as shown, but may also be in ionic or salt form as defined above.
    In highly preferred embodiments, AcHFA is represented by structure 3:
    where 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-13hydroxyoctadec-9-enoic acid (3a), or (cis or trans; RR, SS, RS, or SR) 13 acetoxy-12-hydroxyoctadec -9-enoic acid (3b):
    BE2019 / 5600
    AcHFA has been found to occur naturally in beer because it is formed during fermentation by the yeast enzyme acetyl transferase (ATF).
    An AcHFA precursor is a dihydroxy fatty acid, such as, for example, structures defined above 1-3, wherein the AcHFA precursor has an OH group at any location where there is an OAc group in AcHFA. The yeast ATF enzyme and acetylate (at least) one OH group, to convert the AcHFA precursor to AcHFA.
    Moreover, it has been found that beer with a reduced AcHFA content, compared to the same regular beer, has improved foaming properties. Specifically, reducing the amount of AcHFA increases the stability of beer foam.
    In the present context, the stability of beer foam is measured in accordance with the standards set by the NIBEM Foundation, and expressed in seconds ([s]). Thus beer foam stability indicates the amount of time in seconds that the foam layer is stable under standardized test conditions.
    The NIBEM methodology is well known and can be found as EBC analysis method 9.42.1. The test is performed at 20 ° C at atmospheric pressure. Foam stability is measured as the time between the preparation of the foam under standardized conditions (including a period in which the foam is allowed to drain) until the foam layer has decreased 3 cm in height.
    Beer can refer to any type of beer, including but not limited to ale, porter, stout, lager and bock beer. Beer is preferably a malt-based beer, that is, a beer prepared from the fermentation of wort
    BE2019 / 5600 prepared from malt. Preferably, beer is lager, which is a beer obtained by fermentation at 7-15 ° C using a bottom yeast, and subsequent storage at low temperature. Lager, for example, includes pilsner. Most preferably, a beer as described herein is a pilsner. A pilsner is a light lager.
    In the present context, beer is understood in a broad sense, and includes both regular (alcoholic) beer and low or zero alcohol ('NA') beer. Thus, beer in the present context is preferably beer with an ethanol content of 0-15% by volume ('ABV'), preferably 1-15% by volume. Beer may have been fortified with hops ("hopped beer"), or it may not have been hopped ("unhopped beer"). Hopped beer includes beer prepared using modified hops such as Rho hops.
    In one preferred embodiment, the beer is a regular beer. "Regular beer", in this context, is regular brewed beer, obtained using a fermentation process that results in more than 1% by volume of ethanol. Thus, regular beer, as defined herein, has an ethanol content of greater than 1% by volume, and preferably less than 15% by volume. The ethanol content of a 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, as described above, most preferably a pilsner. Those skilled in the art are capable of obtaining regular beer, including regular lager and pilsner, for example, by the methods described in The Brewer's Handbook (second edition) by Ted Goldammer (2008, Apex Publishers), or by the methods disclosed herein. Alternatively, regular beer can be obtained commercially.
    In another preferred embodiment, the beer is a zero or low alcohol beer ("NA beer"). In the present text, a "zero or low alcohol beer" is a beer with an ethanol content of 1.0% by volume ("ABV") or less, preferably 0.5% by volume or less, more preferably 0, 2 vol.% Or less. Thus NA beer is a beer with an ethanol content of 0 - 1.0% by volume,
    BE2019 / 5600 such as preferably O - 0.5% by volume.
    For example, an NA beer can be obtained by de-alcoholization of regular beer (a "de-alcoholized beer"), or by limited ethanol fermentation of wort (a "beer with limited fermentation").
    One method of obtaining an NA beer as defined herein is by subjecting a regularly brewed beer to a de-alcoholization step, such as, for example, a correction step, a reverse osmosis step, a dialysis step, or a freeze concentration step, to remove ethanol from the fermented beer. These techniques are described, for example, in Brânyik et al, J. Food. Spooky. 108 (2012) 493-506, or in Mangindaan et al, Trends in Food Science & Technology 71 (2018) 3645.
    Another method of obtaining NA beer is to make beer by a limited fermentation process, which yields a beer with limited fermentation. A limited fermentation beer is another type of NA beer as defined herein.
    A beer with limited fermentation is defined as a fermented beer obtained by limited ethanol fermentation of wort. Limited ethanol fermentation of wort is fermentation that does not result in significant net ethanol formation, that is, limited fermentation as defined herein results in 1 vol% or less, preferably 0.5 vol% or less ethanol, more preferably 0, 2 vol.% Or less. Thus, a beer with limited fermentation has an ethanol content of 1.0% by volume or less, preferably 0.5% by volume or less, more preferably 0.2% by volume or less.
    Limited wort fermentation is a process in which the product obtained directly from the fermentation has an ethanol content of 1.0 vol% or less, preferably 0.5 vol% or less, more preferably 0.2 vol% or less . Those skilled in the art are aware of various limited fermentation techniques that do not result in a significant net
    BE2019 / 5600 ethanol formation. Examples are limited ethanol fermentation of wort characterized by • a temperature below 7 ° C, preferably -1 - 4 ° C, such as -0.5 - 2.5 ° C, preferably over a period of 8 - 72 hours, with more preferably 12 - 48 hours ('cold-contact fermented beer'); and / or • a short (eg less than 2 hours) fermentation time, which fermentation quickly stopped due to temperature inactivation, such as by rapid cooling to -0.5 - 1 ° C, optionally followed by subsequent pasteurization ('beer with stopped fermentation' ); and / or • fermentation by a yeast strain producing low amounts of ethanol under the fermentation conditions employed, such as, for example, a yeast strain producing less than 0.2 g ethanol per gram of fermentable sugar in the wort, preferably less than 0.1 g ethanol per gram fermentable sugar. Suitable strains (e.g. Crabtrenegative strains) are known in the art and the amount of ethanol produced under varying fermentation conditions can be determined by routine experiments ("yeast-limited beer"); and / or • fermentation using a first ethanol-producing yeast strain, in the presence of a sufficient amount of a second yeast strain that consumes ethanol, such as Saccharomyces rouxii, to consume substantially all ethanol produced by the first yeast strain; and / or • wort with a content of fermentable sugars, such that a maximum of 1.0% by volume of alcohol is produced upon completion of its fermentation. In this case, the wort usually has a fermentable sugar content of less than 17.5 g / l, preferably less than 12 g / l, more preferably less than 8 g / l ("low sugar worthier").
    A beer with limited fermentation has not been subjected to a deal alcoholization step to the said ethanol content of 1.0% by volume or less, preferably 0.5% by volume or less, more preferably 0.2% by volume or
    BE2019 / 5600 less to achieve. The skilled artisan knows several suitable techniques for de-alcoholization of a fermented beer (see above, with reference to Brânyik et al and Mangindaan et al), and none of these techniques has been used to achieve the said ethanol content. However, a beer with limited fermentation in the present context may optionally be subjected to a deal alcoholization step to reduce the ethanol content of said 1.0 vol% or less, preferably 0.5 vol% or less, more preferably 0, 2% by volume or less, as obtained from the fermentation, to a further reduced ethanol content. Preferably, however, a beer with limited fermentation as defined herein is not subject to a deal alcoholization step at all.
    A beer with limited fermentation in the present context is preferably a sugar-poor beer, a yeast-limited beer, a beer with stopped fermentation, or a cold-contact fermented beer. A beer with limited fermentation in the present context is preferably a cold-contact fermented beer.
    In very preferred embodiments, a beer in the present context is a regular beer, a beer with limited fermentation, or a mixture of the two types of beer, preferably a regular beer, a cold-contact fermented beer, or a mixture of the two types beer. Most preferably, in the present context, the beer is regular beer, with an ethanol content of 1-15% by volume as defined above.
    In order to take full advantage of the increased foam stability, a beer according to the invention preferably has a low sugar content. The total sugar content of a beer according to the invention, defined as the total glucose, fructose, sucrose, maltose and maltotriose, is preferably 0.05 - 5 g / 100 ml, more preferably 0.1 - 1 g / 100 ml even more preferably 0.15-0.5 g / 100 ml. This is because it has been found that in beer with relatively low sugar content
    BE2019 / 5600 foam stabilizing effect of reduced AcHFA amounts is stronger.
    For similar reasons, a beer according to the invention preferably has relatively high levels of free amino nitrogen (FAN). FAN contributes to foam stability and beer according to the invention therefore preferably has a FAN content of 50-160 mg / l, preferably 90-140, more preferably 110-135 mg / l.
    Iso-alpha acids also have a positive effect on beer foam stability. Thus, a beer according to the invention preferably has a total of iso-alpha acids of 2 - 55 mg / 1, preferably 5 - 45 mg / 1, more preferably 10 - 30 mg / 1, more preferably 13 - 25 mg / 1. Iso-alpha acids in this context are defined as the soft resin fraction of lupulin, which is produced in female hop cones. Alpha acids include, for example, humulon, cohumulon and adhumulon.
    In order to obtain beer with a reduced amount of AcHFA as defined herein, three options are disclosed: removal of AcHFA from the beer during or after fermentation, avoidance of formation of AcHFA using an ATF-deficient yeast for fermentation, and removal of an AcHFA precursor from the mixture to be fermented. Thus, the invention discloses a method of increasing the foam stability of beer, comprising a step of fermenting wort to obtain said beer, wherein
    a) the fermentation is accomplished using an acetyl transferase (ATF) arm yeast; and / or
    b) the beer is contacted with an adsorbent capable of adsorbing AcHFA during or after fermentation; and / or
    c) the wort is subjected to a step of removing an AcHFA precursor by an adsorbent capable of adsorbing the AcHFA precursor.
    BE2019 / 5600
    ATF deficient yeast
    In one preferred embodiment, fermentation is accomplished using an acetyl transferase (ATF) deficient yeast (including any ATF ortholog), which yeast is preferably S '. cerevisiae. Such a yeast can be obtained by genetic modification of yeast (preferably S. cerevisiae), such as by replacement or disruption of the ATF-encoding genes (knockout). In the present context, an ATF-deficient yeast means that in yeast types where multiple copies of an ATF gene occur, preferably all copies of that ATF gene have been eliminated [knock out].
    There are two genes associated with acetyl transferase activity: ATF-1 and ATF-2 (in S. Cerevisiae). The ATF-1 gene has gene identification number 854559 (NCBI database) and the ATF-2 gene has gene identification number 853088 (NCBI database). In other yeast types suitable for fermentation, orthologous ATF genes can be located using common general knowledge. As mentioned, in an ATF-1 deficient yeast, preferably all copies of the ATF-1 gene are eliminated. Preferably, all copies of the ATF-2 gene are eliminated in an ATF-2 deficient yeast.
    In one embodiment, the ATF deficient yeast is an ATF-1 deficient yeast. ATF-1 deficiency results in about a 50-90%, preferably about a 65-85% reduction in AcHFA formation. In another embodiment, the ATF deficient yeast is an ATF-2 deficient yeast. ATF-2 deficiency results in about 1-40, preferably about 10-30% reduction in AcHFA formation. In preferred embodiments, the ATF deficient yeast is at least ATF-1 deficient, and preferably also ATF-2 deficient. In very preferred embodiments, the ATF deficient yeast is both an ATF-1 and ATF-2 deficient yeast. Use of a yeast deficient in both ATF-1 and ATF-2 results in complete prevention of AcHFA formation during fermentation.
    BE2019 / 5600
    An ATF deficient yeast can be obtained by well known methods, such as by mutagenesis, preferably random mutagenesis, or by genetic engineering.
    "Mutagenesis" as used herein refers to a process by which at least one mutation is produced in the DNA of at least one yeast cell or spore thereof, such that the genetic information of the yeast cell (s) or spore (s) is altered. Thus, mutagenesis can result in an ATF deficient yeast as described above.
    As used herein, the term "mutation" refers to any change in the DNA of a yeast cell or spore and includes, but is not limited to, point mutation, insertion or deletion of one or more nucleotides, substitution of one or more nucleotides, reading shift mutation and single-stranded or double-stranded DNA break, such as a chromosome break or subtelomer break, and any combination thereof. Preferably, the mutation is located in an ATF gene to result in an ATF deficient yeast as described above.
    Mutagenesis can be performed using any method known in the art, including conventional random mutagenesis methods, such as radiation and chemical treatment, and recombinant DNA technologies, such as site-directed mutagenesis or targeted mutagenesis. Therefore, in one embodiment, the at least one yeast cell or spore of at least the first yeast is subjected to treatment with UV irradiation, X-ray irradiation, gamma irradiation or a mutagen, or genetic engineering.
    "Genetic engineering" is well known in the art and refers to altering the yeast cell or spore genome using biotechnology, thereby introducing a modification of the yeast cell or spore DNA.
    Random mutagenesis refers to mutagenesis techniques that make the exact site of mutation unpredictable and occurs throughout the
    BE2019 / 5600 chromosome of the yeast cell (s) or spore (s) may occur. Usually, these methods involve the use of chemical agents or radiation to induce at least one mutation. Random mutagenesis can be further accomplished using error-prone POR, POR being performed under conditions where the copying precision of the DNA polymerase is low, resulting in a relatively high percentage of mutations in the PCR product. Site-directed mutagenesis can be accomplished using oligonucleotide-directed mutagenesis to yield site-specific mutations in a DNA sequence of interest. Targeted mutagenesis refers to mutagenesis methods that modify a specific or targeted gene in vivo, resulting in a change in the genetic structure targeted at a specific site, such as programmable RNA-guided nucleases, such as TALEN, CRISPR-Cas, zinc finger nuclease or meganuclease technology.
    In a preferred embodiment, mutagenesis in the method of the invention is performed by subjecting the at least one yeast cell or spore to radiation treatment, such as UV irradiation, X-ray irradiation, gamma irradiation, or a mutagenic agent, preferably a chemical agent such as NTG ( N-methyl-N'-nitro-N-nitrosoguanidine) or EMS (ethyl methanesulfonate).
    Adsorption of AcHFA or an AcHFA precursor
    In a further preferred embodiment, the fermented beer is contacted with an adsorbent capable of adsorbing AcHFA. In addition, it is possible to remove the AcHFA precursor from the wort prior to fermentation by contacting the wort with an adsorbent capable of adsorbing the AcHFA precursor.
    It has been found that the same adsorbents can be used to remove the AcHFA precursor from wort and to remove AcHFA from the beer during or after fermentation. That wants to
    BE2019 / 5600 say, the AcHFA precursor and AcHFA themselves adsorb to substantially the same adsorbents. Suitable adsorbents include activated carbon, a hydrophobic adsorbent, a hydrophilic adsorbent and a zeolite. Preferably these adsorbents are used during or after fermentation (preferably after fermentation) to adsorb AcHFA from the beer.
    To improve the foam stability of unhopped beer, the beer can be contacted with any adsorbent suitable for adsorbing AcHFA (or a precursor therefor). Therefore, in preferred embodiments, the beer is unhopped. Optionally, beer can be hopped later, in order to achieve a further foam stability improvement and further (taste) benefits. Hopped beer includes iso-alpha acids, which have a further stabilizing effect on beer foam.
    To improve the foam stability of hopped beer, the beer is preferably contacted with an adsorbent which does not substantially adsorb iso-alpha acids. Iso-alpha acids are derived from hops through wort boiling and are known to have a foam stabilizing effect. The person skilled in the art realizes that it is also possible to use any adsorbent for hopped beer, as long as the adsorbent has a net improving effect on foam stability. This can depend on the amount of hops used and / or the amount of AcHFA formed during fermentation. In preferred embodiments, a hopped beer can be further hopped after the AcHFA removal step is completed.
    Activated carbon is well known in the art and can be used as an adsorbent to remove AcHFA and obtain an AcHFA-reduced beer of the invention. A drawback of activated carbon, however, is that it is not very efficient in AcHFA removal. In addition, activated charcoal adsorbs iso-alpha acids significantly, which is a drawback in the treatment of hopped beer. A further drawback is that activated carbon is the beer
    BE2019 / 5600 discolours, resulting in an overly light drink that is not attractive to consumers.
    Hydrophobic adsorbents include hydrophobic polymeric adsorbents, preferably polymeric adsorbents comprising aromatic and / or acyl groups. Those skilled in the art can easily determine, based on common knowledge of the art and the methods described herein, which adsorbents are suitable for reducing AcHFA (or its precursor) from beer (or wort). It is an advantage of hydrophobic polymeric adsorbents that they are very efficient in removing AcHFA (or an AcHFA precursor). Moreover, the color of the beer is retained and these adsorbents adsorb relatively little, if not none, iso-alpha acids. Accordingly, hydrophobic polymeric adsorbents are preferred, such as, in particular, polystyrene / divinylbenzene (PS / DVB), e.g. PLRP-S, marketed by Agilent.
    Hydrophilic adsorbents are preferably hydrophilic polymeric adsorbents. A very preferred hydrophilic adsorbent is a mixed cellulose ester adsorbent, for example MCE (MF-Millipore ™ membrane filters, Merck). This is the most efficient adsorbent among the adsorbents tested, which leaves the color of the beer intact and does not adsorb iso-alpha acids. In addition, it can be regenerated, which in particular for industrial beer production leads to reduced production costs and less waste material.
    Further preferred adsorbents are, for example, Supelclean ™ adsorbents, such as 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, with the most preferred Supelclean LC C18, Supelclean LC C8. These adsorbents are preferred for the same reasons as described above for MCE and PLRP-S.
    BE2019 / 5600
    A zeolite is, in the present context, preferably a hydrophobic zeolite. This is a silicate-based molecular sieve containing S1O2 and AI2O3 in a molar ratio (SiCYAhOe) of at least 15. The term "molecular sieve" as used herein refers to a microporous material having pores with a diameter of no more than 2 nm. The term "silicate-based" means that the material contains at least 67 wt% silicate. Thus, a "zeolite" is a microporous aluminosilicate. Zeolite adsorbents in the present context can be naturally occurring zeolites or synthetic zeolites.
    It should be understood that hydrophobic silicate-based molecular sieves containing S1O2 and not AI2O3 meet the condition that the molecular sieve contains S1O2 and AI2O3 in the molar ratio of at least 15 (in which case the zeolite is a microporous silicate a microporous alumino-silicate).
    In a preferred embodiment, the adsorbent is a hydrophobic zeolite. The hydrophobic zeolite used in the present process preferably has a SiCL / AhOe molar ratio of at least 40, more preferably of at least 100, even more preferably of at least 200, most preferably of at least 250.
    The average pore size diameter of the hydrophobic zeolite is preferably in the range of 0.2-1.2 nanometer, more preferably 0.3-1.0 nanometer, even more preferably 0.4-0.8 nanometer, and most preferred from 0.45-0.70 nanometer. The pore size diameter of the hydrophobic zeolite can be determined by analyzing the nitrogen adsorption isotherms at 77 K using the t-plot-De Boer method.
    The surface of the hydrophobic zeolite is preferably at least 100 m 2 / g, more preferably 150 to 2000 m 2 / g, and most preferably 200 to 1000 m 2 / g. The area of the hydrophobic molecular sieve can be determined by the BET method.
    BE2019 / 5600
    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 particulate hydrophobic molecular sieve can be determined using a set of seven of different mesh sizes.
    The hydrophobic zeolite is preferably selected from ZMS-5 zeolite, type Y zeolite, zeolite beta, silicalite, ferrierite with only silica, mordenite, and combinations thereof. More preferably, the hydrophobic zeolite is selected from ZMS-5 zeolite, type Y zeolite, zeolite beta, and combinations thereof. Most preferably, the hydrophobic zeolite is ZMS-5 zeolite.
    Sorbents for use as disclosed herein may be used as known in the art.
    For removal of AcHFA during fermentation, the fermentation mixture may be contacted with the adsorbent, for example, by passing the mixture through a column packed with the adsorbent, or by adding the adsorbent as a particulate material to the fermentation mixture and then removing the adsorbent by filtration, cyclone separation or other suitable technique.
    For removal of AcHFA after fermentation, the beer obtained from the fermentation can be contacted with the adsorbent, for example by passing the beer over a column packed with the adsorbent, or by adding the adsorbent as a particulate material to the beer and then removing the adsorbent by filtration, cyclone separation or other suitable technique. In preferred embodiments, the beer obtained from fermentation is introduced into a holding tank where it is contacted with the adsorbent for a period of
    BE2019 / 5600 for example 20 minutes - 24 hours, preferably 0.5 - 5 hours, to adsorb AcHFA. The AcHFA can then be removed by filtration. Alternatively, the beer can be continuously filtered through a filter loaded with the adsorbent.
    For removal of an AcHFA p recursor, the wort may be contacted with the adsorbent prior to fermentation, for example, by passing the wort over a column packed with the adsorbent, or by adding the adsorbent as a particulate material to the wort and subsequent removal of the adsorbent by filtration, cyclone separation or other suitable technique. Preferably, wort is contacted as sweet wort prior to the addition of seed yeast.
    Those skilled in the art know numerous techniques for adsorbing an undesirable component from a liquid mixture by adsorption, and any technique can be used to obtain beer with a reduced amount of AcHFA as defined herein. Suitable techniques are described, for example, in C. Judson King, Separation Processes (2nd edition) McGraw-Hill, Inc. 1980.
    The wort, fermentation mixture or beer is contacted with the adsorbent for a period of time sufficient to adsorb AcHFA or its precursor. This can be easily determined by those skilled in the art based on common knowledge of the art and the methods disclosed herein. Preferably, the contact time is 5 minutes - 48 hours, more preferably 0.5 - 24 hours, more preferably 1 - 20 hours.
    The amount of adsorbent to be used can also be easily determined by one skilled in the art based on common knowledge of the art and the methods disclosed herein. Preferably, the adsorbent is used at a dose of 0.001-10 g / l, more preferably 0.01-5 g / l, more preferably at a dose of 0.05-2.5 g / l.
    BE2019 / 5600
    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 significantly reduced, as can be easily verified by those 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 higher. In preferred embodiments in which AcHFA is removed from beer by adsorption, the amount of AcHFA is reduced to less than 60% of the initial amount, more preferably to less than 20% of the initial amount, most preferably to less than 5% of the initial amount.
    The effect of AcHFA reduction is to increase foam stability by at least 10 seconds, preferably at least 15 seconds, more preferably at least 20 seconds. Foam stability can be increased to 40 seconds, or even up to 50 seconds or higher.
    In highly preferred embodiments, the invention relates to a method for increasing the foam stability of beer, comprising contacting a beer obtained by fermentation with an adsorbent capable of adsorbing AcHFA. The adsorbent and the method of contacting the beer with the adsorbent are as defined above. This results in a beer with increased foam stability, as described above.
    For the purpose of clarity and a brief description, features are described herein as part of the same or separate embodiments; however, it will be appreciated that the scope of the invention may include embodiments with combinations of all or some of the features described.
    The invention will now be further illustrated with the following non-limiting examples.
    BE2019 / 5600
    Methods
    AcHFA analysis
    The AcHFA levels 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 consisted of:
    A: Milli-Q water + 0.1% (v / v) formic acid
    B: acetonitrile + 0.1% (v / v) formic acid
    The analytical column was a 15 cm * 2.1 mm ID UPLC BEH C18 (1.7 µm) column. AcHFA was separated from the other matrix constituents by the following gradient:
    T 0 min: 95% A, 5% B
    T 13 min: 70% A, 30% B
    T 17 min: 5% A, 95% B
    T 25 min: 95% A, 5% B
    Flow rate was adjusted to 0.25 ml / min and the column was kept at 50 ° C with a thermostat. The UPLC system was connected to the MS via an electrospray interface (ESI) operated in negative ion mode. MRM data were recorded for the following transition: m / z 355> 295. Cone voltage was set to 40 volts. Collision energy was set at 15 eV. AcHFA was detected as the compound represented by Structure 3. AcHFA amount was expressed in arbitrary units [arbitrary units] (a.u) obtained from the peak area of the chromatogram to provide quantitative results.
    Determination of sugar content in beer
    Sugar content was measured by Ultra Performance Liquid Chromatography (UPLC). UPLC can conveniently be performed at a temperature of 65 ° C. A suitable choice for the eluent is a mixture of acetonitrile / water, for example in one
    BE2019 / 5600 volume ratio of 75/25. The detector used is typically a refractive index [R1] detector. The sugar content of a sample was determined by comparing the UPLC curve of the sample with calibration curves of standard samples with known sugar concentrations.
    The samples for UPLC were prepared as follows. A beer or wort sample was diluted a factor of 5 by adding acetonitrile / water mixture (50/50 - equal parts by volume). If present, CO2 was removed prior to dilution (e.g., by shaking or stirring the sample). After dilution, the sample was filtered to obtain a clear solution. The filtered sample was injected into the UPLC at 65 ° C using the above eluent.
    Determination of free amino nitrogen (FAN)
    The amount of free amino nitrogen (such as amino acids, small peptides and ammonia) was measured by the Nitrogen by O-Ph thialeddehyde Assay (NOPA) method. The NOPA method was performed using a photometric analyzer (eg, Gallery ™ Plus Photometric Analyzer). According to the NOPA method, a test sample is subjected to treatment with ortho-phthalaldehyde [orthophthalialdehyde] (OPA) and N-acetylcysteine (NAC). This treatment results in the derivatization of primary amino groups present in the test sample to form isoindoles. The content of the isoindoles is then determined using the photometric analyzer at a wavelength of 340 nm. The free amino nitrogen (expressed in mg FAN / 1) can then be calculated based on the measured content of the isoindoles. If necessary, the beer or wort sample may be centrifuged prior to analysis to clarify the sample and / or CO2 removal step (e.g., by stirring or shaking the sample).
    BE2019 / 5600
    Determination of ethanol in beer
    The ethanol content was enjoyed using a photometric analyzer (eg Gallery ™ Plus Photometric Analyzer). The test sample is subjected to an enzymatic method, in which the ethanol contained in the sample is converted into acetaldehyde with alcohol dehydrogenase (ADH). The acetaldehyde content is then determined using the photometric analyzer at a wavelength of 340 nm. The ethanol content can be calculated on the basis of the acetaldehyde content. If necessary, the beer or wort sample is first centrifuged before analysis to clarify the sample and / or subjected to a CO2 removal step (e.g. by stirring or shaking).
    Determination of iso-alpha acids in beer
    Iso-alpha acids were quantitated using EBC 9.47 2010: "Iso-a-acids and reduced iso-a-acids (Rho, Tetra, Hexa) in beer by HPLC."
    Determination of foam stability
    Foam stability is determined in accordance with the standards set by the NIBEM Foundation, EBC 9.42.1.
    Degassing and recarbonation
    All experiments with AcHFA adsorption from beer were performed on regular beer that had been degassed. Beer was carbonated before measuring foam stability.
    Degassed beer was obtained as follows.
    About 200 ml of bottled beer (4 ° C) was carefully transferred (avoid foaming) to clean 500 ml laboratory bottles. The
    BE2019 / 5600 bottles containing the samples were placed in a water bath at 20 ° C and shaken gently (40 rpm) for 40 minutes.
    Then, the shaking intensity was increased to 120 rpm, and shaking was continued for 30 minutes to obtain the degassed beer. Throughout the degassing treatment, the air chamber of the bottles was continuously purged with nitrogen gas to prevent oxidation of the beer.
    Recarbonation was achieved on a lab scale as follows. Millipore stainless steel kegs (model Millipore catalog no. Xx6700p05) were prepared for use as regular beer kegs. Barrels were cleaned and rinsed with CO2 to remove air prior to addition of the beer. Beer samples of about 1 - 2 liters were transferred to the vats and the samples were placed under 2.5 bar CCL pressure for 30 minutes with shaking at 100 rpm. The entire procedure was done at 20 ° C.
    CO2 pressure was used to release beer using a thick needle placed about 0.7 cm from the bottom of the barrel. Thus, foam was prepared by connecting the Nibem foam dispenser instrument to the barrel (under CO2 pressure), similar to what was done using bottled beer. Foam stability was determined according to the Nibem method described.
    Example 1
    Removal of AcHFA from beer results in foam stabilization. Degassed beer was incubated with 0.1 g / l commercial methyl cellulose ester adsorbent (MCE, MF-Milhpore ™ membrane filters, Merck) for 16 hours at room temperature. The beer was then filtered through a 0.2 μm filter and re-carbonated.
    The results show that adsorption of AcHFA to MCE and subsequent removal of AcHFA from the beer results in beer with
    BE2019 / 5600 improved foam stability. The 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 more than 90% of the AcHFA resulted in an increase in foam stability of 17%.
    The AcHFA removal process has been shown to result in efficient removal of AcHFA in both hopped beer and unhopped beer (data not shown). In addition, it has been shown that MCE does not adsorb iso-alpha acids significantly and that the beer color is not reduced. This is beneficial since iso-alpha acid presence has a further stabilizing effect on beer foam. Therefore, MCE is a very preferred adsorbent.
    Example 2
    AcHFA is a foam negative factor
    Degassed regular beer was obtained as described in example 1. The beer in this case was a hop-free, fully malted beer. The degassed beer was incubated with 0.2 g / l PLRP-S (Agilent) at room temperature for 16 hours to effect complete removal of AcHFA and obtain an AcHFA-free, unhopped beer.
    The adsorbent was filtered and isolated, and washed with acetonitrile to obtain crude AcHFA. The crude AcHFA was fractionated using a water-to-acetonitrile gradient in a counterphase HPLC column (C18). The fractions obtained were tested for their effect on foam stability by addition to beer and the foam negative fractions were collected and analyzed by LC-MS and characterized as AcHFA by NMR as defined herein. Thus, pure AcHFA was isolated and characterized.
    BE2019 / 5600
    To degassed hopped and degassed unhopped beer with regular amounts of AcHFA as obtained by conventional fermentation, an additional amount of pure AcHFA was added. 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 shown in Figures 2a and 2b.
    Table 1
    beer foam stability[sec] beer foam stability[sec] hopped beer 271 hop free beer 202 + 0.25 mg / 1 AcHFA 270 + 1.5 mg / 1 AcHFA 198 + 0.5 mg / 1 AcHFA 268 + 2.0 mg / 1 AcHFA 194 + 1.0 mg / 1 AcHFA 263 + 4.0 mg / 1 AcHFA 162 + 2.0 mg / 1 AcHFA 251 + 4.0 mg / 1 AcHFA 233
    The results show that by increasing the amount of AcHFA in regular beer, the foam stability of beer decreases rapidly. Thus, it was concluded that AcHFA is a foam negative factor and that beer with lower amounts of AcHFA has increased foam stability.
    Example 3
    Beer with a reduced amount of AcHFA has higher foam stability. In a further experiment to achieve complete removal of AcHFA, incubation for 16 hours at room temperature with 0.2 g / l PLRP-S (Agilent) AcHFA-free hopped beer and AcHFA- free hop free beer. Prior to AcHFA addition to the hopped beer, loss of iso-alpha acids during the adsorption step was compensated
    BE2019 / 5600 by adding 20 mg / 1 iso-alpha acids [iso-alpha acids] ('IAA', B-hop, Hopsteiner). Both AcHFA beers were then degassed.
    AcHFA-free beers were added in varying amounts of AcHFA. After gentle homogenization and recarbonation as described above, foam stability was determined. The results are shown in Table 2 and in Figures 3a and 3b.
    Table 2
    beer foam-stability beer foam-stability Hopped beer (AcHFA-free) 261 Hop free (regular AcHFA) 200 Hopped beer (+ IAA), AcHFA free 317 Hop free (AcHFA free) 261 Hopped beer (+ IAA) + 0.25 mg / 1 AcHFA 306 Hop free + 0.25 mg / 1 AcHFA 251 Hopped beer (+ IAA) + 0.5 mg / 1 AcHFA 298 Hop free + 0.5 mg / 1 AcHFA 234 Hopped beer (+ IAA) + 0.75 mg / 1 AcHFA 295 Hop free + 0.75 mg / 1 AcHFA 227 Hopped beer (+ IAA) + 1.0 mg / 1 AcHFA 285 Hop free + 1.0 mg / 1 AcHFA 221 Hopped beer (+ IAA) + 1.5 mg / 1 AcHFA 273 Hop-free + 1.5 mg / 1 AcHFA 214
    The results show that decreasing the amount of AcHFA in beer below levels in regular beer results in an increase in foam stability in an AcHFA concentration dependent manner. Beer without AcHFA has the highest foam stability.
    Example 4
    Reduce AcHFA in beer
    A regular (hop-free) beer with a conventional amount of AcHFA was obtained. This beer was degassed as described and contacted in 30 ml aliquots (10 column volumes) in a 3 ml SPE column with the same amount of various Supelclean ™ adsorbents (Sigma Aldrich) and then re-carbonated. The results are shown in Table 3 and Figure 4.
    BE2019 / 5600
    Table 3
    sample AcHFA (please) % from start Hop free blank 870 100% + LC WCX 815 94% + LC Si 815 94% + LC C18 25 3% + LC C8 25 3% + LC Ph 140 16% + LC CN 330 38% + LC SAX 680 78% + LC SCX 470 54%
    The results show that a variety of hydrophobic and hydrophilic adsorbents are capable of reducing the amount of AcHFA in beer. Preferred adsorbents are LC C18, LC C8 and LcPh. In preferred embodiments, AcHFA is reduced to less than 60% of the initial amount, more preferably to less than 20% of the initial amount, even more preferably to less than 5% of the initial amount.
    Example 5
    Reduction of AcHFA in beer using PLRP-S and activated carbon
    Hop-free, degassed beer was contacted with PLRP-S (0.1 mg / l) and 1.0 mg / l activated carbon (Aktivkohle, art. 2186 Merck) and then recarbonated. The results are shown in Figure 5.
    The results show that PLRP-S is more efficient at removing AcHFA from beer. In addition, it has been observed that activated carbon removes color from the beer, which is considered a drawback. PLRP-S leaves the beer color intact. In addition, PLRP-S does not adsorb iso-alpha acids
    BE2019 / 5600 significant. Therefore, PLRP-S and MCE (see Example 1) are preferred adsorbents over activated carbon.
    Example 6
    Removal of AcHFA precursor from sweet wort to obtain beer with a reduced amount of AcHFA over conventional beer
    In a conventional pilot plant brewing process, sweet wort was treated with PLRP-S by coating 750 g of PLRP-S on a PVPP filter installed between the clarification vessel and the wort kettle, and treating 15 hl of sweet wort with said filter prior to entering the wort kettle (figure 6). Beer was then brewed in a conventional manner (hop addition, boiling, swirling, fermentation and after-treatment and bottling).
    A similar beer was brewed from the same wort using the same process conditions, but without filtering the sweet wort over the filter loaded with PLRP-S.
    Analysis of the amount of the AcHFA precursor (a dihydroxy fatty acid) was done on a sample of the hopped wort (wort to which the seed yeast is added), using HPLC conditions as described for AcHFA. The amount of AcHFA was determined on the final beer. The results are shown in Table 4.
    Blank Experimental foam stability (sec) 270 288 total iso-a-acids (mg / 1) 18.4 18 AcHFA precursor in wort (a.u.) 22900 6530 AcHFA in beer (please) 25900 5790
    BE2019 / 5600
    The results show that removal of the AcHFA precursor by adsorption can prevent formation of AcHFA in the final beer. As a result, the foam stability of the beer obtained increases.
    The results further demonstrate that the adsorption process can be applied on an industrially relevant scale.
    Example 7
    Obtaining beer with a reduced amount of AcHFA by yeast modification
    In this example, using CRISPR-Cas9 technology, a deletion library of S 'became. pastorian wild-type (WT) yeast constructed with different combinations of deleted alleles of ATF1 and ATF2. S. pastorianus-VTV contains five copies of ATF1 and four copies of ATF2 from both S. cerevisiae and S. eubayanus genomes. With the CRISPR-Cas9 system functional in S. pastorianus, all copies of one gene are targeted in a single transformation. Ultimately, nine ATF1 / 2 genes from four different chromosomes were deleted in just two transformations. The ATF-1 deficient yeast is called 'strain a'. The ATF-2 deficient yeast is called 'strain b'. The yeast deficient in both ATF-1 and ATF-2 is called 'strain c'. The effect of different knockouts in the was studied by culturing the strains under brewing conditions. The strains showed comparable growth rates, sugar consumption profiles and ethanol production during a standard brewing fermentation in 15 ° Plato wort.
    Obtaining mutants from wild type yeast
    Mutant strains were obtained following the methodology described in 'CRISPR Cas9 mediated gene deletions in lower yeast Saccharomyces pastorianus', Arthur R. Gorter de Vries et al., Microb Cell
    BE2019 / 5600
    Fact (2017) 16: 222. A representation of the mutations is shown in Figure 7a.
    Characterization of WT with ATF deletions under brewing conditions
    To check for fermentation performance, the strains were inoculated into 15 ° Plato autoclaved and filter sterilized wort with additional zinc to meet yeast growth requirements. Three bottles with the same cell concentration of approximately 5 million cells were inoculated per strain. All bottles were sampled for analysis on the HPLC at the beginning of the experiment and after seven days of fermentation. However, one bottle of each triplicate was also sampled during the fermentation to monitor the sugar consumption and the production of glycerol, ethanol and biomass, to compare with fermentation with WT. After fermentation, the supernatant from each bottle was analyzed by HPLC for sugar, glycerol and ethanol. It was observed that the mutant strains when used in a fermentation with respect to the wild type yeast exhibit essentially identical behavior. The sugar consumption profile over time for glucose, maltose, maltotriose and fructose indicates that a substantially identical consumption profile is used. Also glycerol and ethanol production as monitored during the fermentation was essentially identical. The cell growth rate (as measured by optical density) was also essentially identical. An exemplary figure showing the ethanol production of strains a, b and c is inserted as Figure 7b. On all parameters tested, the mutant strains showed essentially identical characteristics and a substantially identical time course. The mutant strains could therefore be used in existing production processes to reduce the amount of AcHFA in the final beer.
    Thus, ATF-deficient yeast types were obtained which could be used 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
    BE2019 / 5600 deficient in both ATF-1 and ATF-2 (strain c) were used in a standard wort fermentation to assess their activity in a standard beer brewing process. The different yeasts - 5 mio cells / ml inoculated - were fermented at 150 ° C for 7 days under microaerobic conditions in 150 ml fully malted 15-16 ° P wort in 250 ml infusion bottles, shaken at 200 rpm. The beer obtained was compared to beer obtained with the unmodified WT yeast. The results are shown in Table 5:
    Table 5:
    yeast type AcHFA (please) % relative to WT WT wild type (WT) 28200 100 strain a deletion ATF 1 4155 15 trunk b deletion ATF2 21800 77 strain c deletion ATF1 + ATF2 not quite 0
    The results show that using an ATF-1 deficient yeast, AcHFA is reduced to 15% of the amount of AcHFA observed using a wild type yeast. With an ATF-2 deficient yeast, AcHFA is reduced to 77% of the amount of AcHFA observed using a wild type yeast. Using a yeast deficient in both ATF-1 and ATF-2 (preferably all copies), AcHFA formation can be completely suppressed.
    Example 8
    Removal of AcHFA from regular beer on an industrially relevant scale to obtain beer with a reduced amount of AcHFA
    Beer was brewed in a pilot plant using a conventional process and a conventional yeast. The brewing process resulted in lager beer. Beer with added iso-alpha acids ('IAA') was added.
    BE2019 / 5600 (20 mg / 1 B-hop, Hopsteiner), as well as beer without added isoalfa acids.
    The lager was filtered and stabilized with PVPP, and stored in a holding tank, where it was contacted with 0.1 g / l PLRP-S (Agilent) for one or two hours. The beer was then filtered to remove the PLRP-S and the adsorbed AcHFA (Figure 8a). Alternatively, the beer obtained from fermentation could be filtered through a PLRPS coated filter to remove AcHFA (Figure 8b; data for the beer obtained not shown, but comparable to beer obtained by the process shown in Figure 8a).
    AcHFA amounts were analyzed and compared to the amount in beer without the mentioned filtration step. In addition, the foam stability of the beer obtained was compared.
    The results show that by treating beer as obtained from fermentation with PLRP-S for one or two hours, the AcHFA amount could be significantly reduced. This had a significant and positive effect on foam stability, both in beer with and without added isoalfa acids. Therefore, adsorbents such as PLRP-S can be used to remove AcHFA and increase foam stability of beer.
    权利要求:
    Claims (11)
    [1]
    Conclusions
    A beer comprising less than 2 mg / l AcHFA, wherein AcHFA is a C12 - C22 fatty acid, comprising a carboxylic acid group and a C11 C21 linear alkyl group, which alkyl group may be partially unsaturated, and which alkyl group is substituted with at least one hydroxyl group and at least one acetate group.
    [2]
    A beer according to claim 1, wherein AcHFA is defined by structure 1

    [3]
    A beer according to claim 1 or 2, wherein AcHFA is defined by structure 2:
    BE2019 / 5600
    a

    [4]
    A beer according to any one of claims 1 to 3, comprising 0-15% by volume of ethanol.
    [5]
    A beer according to any one of claims 1 to 4, comprising less than 1.5 mg / l AcHFA, preferably less than 1.0 mg / 1, more preferably less than 0.5 mg / 1, even more preferably less than 0.25 mg / 1 1.
    [6]
    A beer according to any one of claims 1 to 5, comprising a total of iso-alpha acids of 5-55 mg / l.
    [7]
    A beer according to any one of claims 1 to 5, comprising a total sugar content, defined as the total of glucose, fructose, sucrose, maltose, and maltotriose, of 0.05-5 g / 100 ml.
    [8]
    A method of increasing the foam stability of beer, comprising a step of fermenting wort to obtain said beer, wherein
    a) the fermentation is achieved by using an acetyl transferase (ATF) deficient yeast; and / or
    b) the beer is contacted with an adsorbent capable of adsorbing AcHFA during or after the fermentation; and / or
    c) the wort is subjected to a step of removing an AcHFA precursor by an adsorbent capable of adsorbing the AcHFA precursor.
    [9]
    A method according to claim 8, wherein the adsorbent is an activated carbon, a hydrophobic adsorbent, a hydrophilic adsorbent, or a zeolite.
    BE2019 / 5600
    [10]
    A method according to claim 8 or 9, wherein the adsorbent is an activated carbon, a polystyrene / divinylbenzene adsorbent, a mixed cellulose ester adsorbent, or a hydrophobic zeolite, preferably a polystyrene / divinylbenzene
    5 adsorbent, a mixed cellulose ester adsorbent, or a hydrophobic zeolite.
    [11]
    A method according to any of claims 8-10, comprising contacting a beer obtained by fermentation with an adsorbent, preferably a polystyrene / divinylbenzene
    Adsorbent, a mixed cellulose ester adsorbent, or a hydrophobic zeolite capable of adsorbing AcHFA.
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    同族专利:
    公开号 | 公开日
    EP3850076A1|2021-07-21|
    WO2020055235A1|2020-03-19|
    BE1026596A9|2020-06-03|
    BE1026596B9|2020-06-10|
    CA3108471A1|2020-03-19|
    AR116382A1|2021-04-28|
    BE1026596A1|2020-04-01|
    CO2021003787A2|2021-04-08|
    BR112021004431A2|2021-06-22|
    JP2022512543A|2022-02-07|
    NL2023802B1|2020-05-01|
    AU2018441502A1|2021-02-25|
    CN112673083A|2021-04-16|
    US20210309950A1|2021-10-07|
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    法律状态:
    2020-05-29| FG| Patent granted|Effective date: 20200406 |
    优先权:
    申请号 | 申请日 | 专利标题
    PCT/NL2018/050588|WO2020055235A1|2018-09-10|2018-09-10|Foam stability|
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