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    • 专利摘要:
    production of low-alcohol or non-alcoholic beer with pichia kluyveri yeast strains. the present invention relates to a beverage with low alcohol content or without alcohol, with a taste profile very close to a beer of at least 4% alcohol (vol/vol), can be produced by using yarn of pichia kluyveri yeast. in particular, pichia kluyveri yeast yarns use only glucose in the wort, and have the ability to convert this substrate into a high concentration of specific flavor compounds, which are normally produced by saccharomyces ssp yeast yarns used for beer fermentation. in this way, pichia kluyveri yeast strands can be used to produce a low-alcohol or non-alcoholic beverage, depending on the glucose levels in the wort. the main flavoring compounds produced by pichia kluyveri in must fermentation are isoamyl acetate, isoamyl alcohol, ethyl butyrate, ethyl hexanoate and ethyl octanoate.
    公开号:BR112015021810B1
    申请号:R112015021810-5
    申请日:2014-03-07
    公开日:2021-06-22
    发明作者:Sofie Saerens；Jan Hendrik Swiegers
    申请人:Chr. Hansen A/S；
    IPC主号:
    专利说明:

    FIELD OF THE INVENTION
    [0001] The present invention relates to the field of preparing a low-alcohol or alcohol-free beverage by fermentation. Specifically, the invention relates to a method of preparing a low-alcohol or non-alcoholic beverage with high levels of flavor compounds derived from desirable fermentation comprising a step of fermenting a wort with a Pichia kluyveri yeast strain to obtain a low-alcohol or non-alcoholic beverage, a low-alcohol or non-alcoholic beverage obtainable by the method, and a low-alcohol or non-alcoholic beverage comprising a high concentration of specific flavor compounds such as isoamyl acetate and ethyl octanoate. BACKGROUND OF THE INVENTION
    [0002] Due to the increased demand for healthier foods and beverages, the reduction of ethanol in alcoholic beverages, especially beer and wine, is of considerable commercial interest.
    [0003] Low-alcohol, non-alcoholic or reduced-alcohol beers (also called light beer, non-alcoholic, non-alcoholic, bland, mild British beer, non-alcoholic beer) are non-alcoholic or low-alcohol beers that aims to reproduce the full flavor of standard beers that normally contain more than 4% (vol/vol) alcohol. Many beers from EU countries with low alcohol content are divided into non-alcoholic beers, which contain no more than 0.5% (vol/vol) alcohol, and low-alcohol beers, which contain no more than 1.2% (vol/vol) of alcohol. In the United States, non-alcoholic beer means that there is no alcohol present, although the maximum limit of 0.5% (vol/vol) of alcohol corresponds to the so-called non-alcoholic or non-alcoholic beer (Montanari et al. 2009).
    [0004] The conversion of must into alcoholic beer is the result of the yeast fermentation process of Saccharomyces ssp. which results in the production of alcohol and fermentation-derived flavor components such as esters (eg isoamyl acetate) and higher alcohols. Isoamyl acetate is an important compound in beer flavor (Saerens et al. 2010). Combining the flavor of raw materials, mainly malt and hops, together with the fermentation process, results in the unique flavor and taste of beers (Swiegers et al. 2007, Saerens et al. 2010).
    [0005] Currently low-alcohol beers are often brewed by producing high-alcohol beer (with an alcohol concentration above 4% (vol/vol)) and then removing the alcohol by physical processes (evaporating alcohol/distillation or reverse osmosis). In the case of some non-alcoholic or non-alcoholic beers, no fermentation takes place and in some cases the beers are produced simply by diluting high-alcohol beer with water.
    [0006] As physical processes result in the evaporation, loss or dilution of fermentation-derived flavor components (or a total absence of fermentation-derived flavor components, as in the case of non-fermented non-alcoholic or alcohol-free beer), these types of beers are often characterized as being less flavorful than high-alcohol beer or may have an undesirable wort-like taste (Zufall and Wackerbauer. 2,000).
    [0007] German patent DD 288619 A5 describes the use of Pichia farinosa yeast strains to ferment hop-free beer wort to obtain a non-alcoholic fruit-flavored drink.
    [0008] However, there is a need for improved methods to prepare a low-alcohol or non-alcoholic fermented beverage, in which the presence of desirable flavor compounds, such as esters and higher alcohols, is accentuated. SUMMARY OF THE INVENTION
    [0009] The present invention is based on the surprising findings of the inventors that Pichia kluyveri yeast strains, which have the ability to ferment only glucose in beer wort, have the ability to produce high concentrations of compounds of desirable flavor only with the production of little ethanol.
    [00010] The brewer's yeast Saccharomyces pastorianus, Saccharomyces carlsbergensis and Saccharomyces cerevisiae use glucose as well as maltose, the main sugar in the wort (Boulton and Quain. 2010) that results in the formation of alcohol and flavor compounds during fermentation. The higher the concentration of wort sugars, the more alcohol and flavor compounds will be produced. To produce less alcohol, a solution is to use a yeast that ferments only glucose and not maltose, thus producing only alcohol from the glucose. Glucose is present at much lower concentrations than maltose in the wort (Table 1) and therefore less alcohol will be produced from the same amount of wort compared to when a conventional brewer's yeast, eg Saccharomyces pastorianus or Saccharomyces carlsbergensis, is used. Such yeast types do exist and examples include certain Saccharomyces and non-Saccharomyces yeast strains, such as Saccharomycodes ssp (Liu et al. 2011).
    [00011] However, only a relatively small amount of flavor will be produced by these yeast strains from the limited amount of glucose, which results in a beer with lower concentrations of important flavor compounds than beers with more than 4% (vol. /vol) of alcohol. TABLE 1. SPECTRUM OF SUGAR TYPICAL OF MUST (STEWART. 2010)

    [00012] One way to solve this problem is to identify a highly aromatic yeast, which does not have the ability to use maltose to produce high concentrations of flavor compounds derived from relevant fermentation of limited amounts of glucose, which results in similar or more concentrations of important flavor compounds than when full fermentation takes place.
    [00013] The inventors of the present invention found that through the use of this method and Pichia kluyveri yeast strains, it is possible to produce non-alcoholic beer and low-alcohol beer (according to EU legislation) with the same flavor as standard beers , high alcohol content. It is not possible to use conventional brewer's yeast, such as Saccharomyces cerevisiae and Saccharomyces pastorianus or Saccharomyces carlsbergensis, dilution, physical alcohol removal processes or beers produced from unfermented substrates.
    [00014] Consequently, a first aspect of the invention relates to a method of preparing a low-alcohol or alcohol-free beverage, comprising the steps: a) providing a must; and b) fermenting the wort with at least one Pichia kluyveri yeast strain to obtain a low-alcohol or non-alcoholic beverage.
    [00015] A further aspect of the present invention relates to a low-alcohol or alcohol-free beverage obtainable by the method, according to the first aspect of the invention.
    [00016] A further aspect of the present invention relates to a low-alcohol or non-alcoholic beverage, wherein the low-alcohol or non-alcoholic beverage has an isoamyl acetate content of at least 0.5 ppm (mg/L ).
    [00017] A still further aspect of the present invention relates to the use of a Pichia kluyveri yeast strain for preparing a low-alcohol or non-alcoholic beverage. BRIEF DESCRIPTION OF THE DRAWINGS
    [00018] Figure 1 illustrates a beer brewing process optimized for Pichia kluyveri beer. Comparison of standard infusion process (left) versus Pichia kluyveri (right). Steps that are different from the standard infusion process are highlighted.
    [00019] Figure 2 illustrates the flavor profile comparison of 5 different non-alcoholic beers: 3 horses. Hollandia, Bavaria (all with 0% alcohol) and beers produced with Pichia kluyveri A strain (0.1% (vol/vol) alcohol) and B strain (0.2% (vol/vol) alcohol). Carlsberg Pilsen beer with 4.6% (vol/vol) alcohol was used as a reference lager and two other lagers are also shown: Stella (5.2% (vol/vol) alcohol) and Heineken ( 5.0% (vol/vol) alcohol). 5 different flavor compounds were measured with headspace-GC-FID: isoamyl acetate, isoamyl alcohol, ethyl butyrate, ethyl hexanoate and ethyl octanoate. The flavor compound concentrations in Carlsberg Lager were used as a reference and was scaled to be 1. The flavor concentrations in all beers were put on the same scale to have the ability to compare values.
    [00020] Figure 3 shows the comparison of flavor profile of 4 different beers: non-alcoholic beer with Pichia kluyveri A strain (0.1% (vol/vol) alcohol), low-alcohol beer with Pichia kluyveri A strain (0.7% (vol/vol) alcohol), Carlsberg lager (4.6% (vol/vol) alcohol) which was used as a reference lager (control) and beer malt (1.8 % (vol/vol) alcohol). 5 different flavor compounds were measured with headspace-GC-FID: isoamyl acetate, isoamyl alcohol, acetate butyrate, ethyl hexanoate and ethyl octanoate. The flavor compound concentrations in the Carlsberg Lager were used as a reference and were scaled to be 1. The flavor concentrations in the 3 beers were placed on the same scale to have the ability to compare values.
    [00021] Figure 4 illustrates the diacetyl concentrations in beer evaluations with Pichia kluyveri A strain and a brewer's yeast Saccharomyces cerevisiae.
    [00022] Figure 5 depicts the proliferation of yeast cells during fermentation.
    [00023] Figure 6 shows isoamyl acetate ethyl decanoate and phenylethyl acetate concentrations in all fermentation products with Saccharomycodes ludwigii and Pichia kluyveri, as well as in the basic wort (with and without hops). w/o = without.
    [00024] Figure 7 illustrates concentrations of butyl acetate and isobutyl acetate in all fermentation products with Saccharomycodes ludwigii and Pichia kluyveri, as well as in the basic wort (with and without hops). w/o = without.
    [00025] Figure 8 shows concentrations of isobutanol, 1-butanol and isoamyl alcohol in all fermentation products with Saccharomycodes ludwigii and Pichia kluyveri, as well as in the basic wort (with and without hops). w/o = without.
    [00026] Figure 9 depicts concentrations of hexanoic, octanoic and decanoic acid in all fermentation products with Saccharomycodes ludwigii and Pichia kluyveri, as well as in the basic wort (with and without hops). w/o = without. DETAILED DESCRIPTION OF THE INVENTION
    [00027] The inventors of the present invention have unexpectedly found that Pichia kluyveri yeast strains have the ability to enhance the presence of desirable flavor compounds in beverages prepared by wort fermentation with Pichia kluyveri yeast strains. In particular, it has been found that concentrations of desirable beer flavor compounds, such as isoamyl acetate, isoamyl alcohol and ethyl octanoate, have been increased.
    [00028] Surprisingly, this accentuation of beer flavor compounds is present when yeast strains from the species Pichia kluyveri are used in the absence of conventional infusion yeast strains, such as yeast strains from the genera Saccharomyces and Brettanomyces.
    [00029] The beer brewing process is well known to a person skilled in the art and can be referred to as follows (see Figure 1); Malt is prepared from dry, germinated cereal grains (mainly barley or wheat) and landfilled in the mill which may contain unmalted adjuncts. The grind is blended (mixed with water and soaked) to allow the enzymes in the malt to convert the starch into sugars. The grain particles and adjuncts are separated from the liquid wort in a process called clarification. The malt manufacturing and crushing steps can be skipped by adding water to the malt extract. After adding hops and/or other ingredients such as herbs and sugars, the wort is boiled (hops can also be added after boiling), cooled and aerated. The wort is then moved to a fermentation tank and fermented by adding an infusion yeast. The first fermentation, which typically lasts 5 to 10 days, can be followed by a second fermentation step using an infusion yeast. After fermentation, the fresh beer or "green" beer is conditioned, optionally filtered and aerated. The beer is filled in bottles and optionally pasteurized.
    [00030] Hops can be added to the wort to balance the malt sweetness with bitterness and give the beer desirable flavors and aromas. There are many varieties of but not limited to Ahtanum, Amarillo, Apollo, Bravo, Calypso, Cascade, Centennial, Chelan, Chinook, Citra, Cluster, Columbus, Comet, Crystal, El Dorado, Eroica, Galena, Glacier, Greenburg, Horizon Liberty, Millenium, Mount Hood, Mount Rainier, Mosaic, Newport, Nugget, Palisade, San Juan, Santiam, Satus, Simcoe, Sonnet Golding, Sterling, Summit, Super Galena, Tillicum, Tomahawk, Ultra, Vanguard, Warrior, Willamette, Zeus, Admiral, Brewer's Gold, Bramling Cross, Bullion, Challenger, First Gold, Fuggles, Goldings, Herald, Northdown, Northern Brewer, Phoenix, Pilgrim, Pilot, Pioneer, Progress, Target, Whitbread Golding Variety (WGV), Hallertau, Hersbrucker , Saaz, Tettnang, Spalt, Ella, Feux-Coeur Francais, Galaxy, Green Bullet, Hallertau Aroma, Kohatu, Motueka, Nelson Sauvin, Pacific Gem, Pacific Jade, Pacifica, Pride of Ringwood, Rakau, Riwaka, Southern Cross, Sticklebract, Summer, Super Alpha, Super Pride, Topaz, Wai-iti, Hallertau Herkules, Hallertau Magnum, Hallertau Taurus, Magnum, Merkur, Opal, Perle, Saphir, Select, Smaragd, Tradition, Bor, Junga, Lublin, Marynka, Premiant, Sladek, Strisselspalt, Styrian Atlas, Styrian Aurora, Styrian Bobek, Styrian Celeia, Styrian Golding, Sybilla, Tardif de Bourgogne and Sorachi Ace.
    [00031] The method for preparing a low-alcohol or alcohol-free beverage of the present invention comprises the steps: a) providing a must; and b) fermenting the wort with at least one Pichia kluyveri yeast strain to obtain a low-alcohol or non-alcoholic beverage.
    [00032] The term "low-alcohol beverage" herein is identified as a drinking liquid with an alcohol content of more than 0.5% (vol/vol) alcohol and no more than 1.2% (vol) /vol) of alcohol.
    [00033] The term "alcohol-free beverage" herein is defined as a drinking liquid with an alcohol content of no more than 0.5% (vol/vol) alcohol.
    [00034] The term "must" in this document has the conventional meaning in the art and refers to a sugary liquid extracted from the crushing process of brewing beer.
    [00035] The concentration of glucose in the wort is critical to the percentage of alcohol resulting from the drink and can be adjusted by modifying the crushing process, by adding enzymes and by other methods known to the person skilled in the art.
    [00036] In a preferred embodiment, the wort has a glucose concentration between 0.01% to 3% (w/vol), such as between 0.5% to 1.0% (w/vol).
    [00037] Preferably, the fermentation in step b) is carried out under sterile conditions (controlled fermentation) and with Pichia kluyveri as the only species of yeast strain added.
    [00038] The skilled person will readily be able to determine an inoculation concentration of the Pichia kluyveri yeast strain suitable for preparing a low-alcohol or non-alcoholic beverage, according to the method.
    [00039] In a preferred embodiment, the Pichia kluyveri yeast strain is inoculated at a concentration of at least 1x104 CFU/ml (colony forming units/ml), such as at least 5x104 CFU/ml, such as at least 1x105 CFU/ ml, such as at least 5x105 CFU/ml, such as at least 1x106 CFU/ml, such as at least 5x106 CFU/ml.
    [00040] In another preferred embodiment, the method further comprises step a1) adding to the wort at least one hop variety before fermentation of the wort with the at least one Pichia kluyveri yeast strain.
    [00041] In another preferred modality, fermentation of the wort with at least one Pichia kluyveri yeast strain is carried out for at least 7 days, such as for at least 14 days, such as for at least 21 days, such as for at least at least 28 days.
    [00042] In another preferred modality the fermentation of the must with at least one Pichia kluyveri yeast strain is carried out at a temperature of between 18°C and 22°C.
    [00043] In yet another preferred embodiment, the method additionally comprises step c) bottling and pasteurizing in a bottle the low-alcohol or non-alcoholic beverage.
    [00044] Preferably, the at least one Pichia kluyveri yeast strain has the ability to ferment glucose but not maltose or maltotriose. This can be tested with the proliferation of the yeast strain in a culture medium with only maltose or maltotriose.
    [00045] In a preferred embodiment, the at least one Pichia kluyveri yeast strain is selected from the group consisting of Pichia kluyveri PK-KR1 (JT1.28; strain A), which was deposited on August 24, 2006 in the National Measurement Institute, 541-65 Clarke Street, South Melbourne, Victoria 3205, Australia, from the University of Auckland, School of Biological Sciences, Auckland 1142, New Zealand, under accession numbers V06/022711 , and Pichia kluyveri PK-KR2 (JT3.71), which was deposited on 24 August 2006 at the National Institute of Measurement, 541-65 Clarke Street, South Melbourne, Victoria 3205, Australia, by the University of Auckland, School of Biological Sciences, Auckland 1142, New Zealand, under accession numbers V06/022712.
    [00046] Preferably, the low-alcohol or non-alcoholic beverage is a low-alcohol or non-alcoholic beer.
    [00047] The term "beer" as used herein refers at least to beers prepared from blends prepared from malted cereals as well as blends prepared from unmalted cereals, and blends prepared from a malted cereal blend and not malted. The term "beer" also refers to beers made with adjuncts and beers with all possible alcohol content.
    [00048] The term "low alcohol beer" herein refers to a beer with an alcohol content of more than 0.5% and no more than 1.2% (vol/vol) alcohol.
    [00049] The term "alcohol-free beer" herein refers to a beer with an alcohol content of no more than 0.5% ABV.
    [00050] A further aspect of the present invention relates to a low-alcohol or alcohol-free beverage obtainable by the method described herein.
    [00051] In a preferred embodiment, the low-alcohol or non-alcoholic beverage is a low-alcohol or non-alcoholic beer.
    [00052] A further aspect of the present invention is directed to a low-alcohol or non-alcoholic beverage, wherein the beverage has an isoamyl acetate concentration of at least 0.5 ppm (parts per million; mg/L), such as at least 0.6 ppm, such as at least 0.7 ppm, such as at least 0.8 ppm, such as at least 0.9 ppm, such as at least 1.0 ppm, such as at least 1.2 ppm, such as at least 1.4 ppm, such as at least 1.6 ppm, such as at least 1.8 ppm, such as at least 2.0 ppm, such as at least 2.5 ppm, such as at least 3.0 ppm, such as at least 3.5 ppm, such as at least 4.0 ppm, such as at least 4.5 ppm, such as at least 5.0 ppm.
    [00053] Preferably, the low-alcohol or non-alcoholic beverage has an ethyl octanoate concentration of at least 10 ppb (parts per billion; μg/L), such as at least 11 ppb, such as at least 12 ppb, such as at least 13 ppb, such as at least 14 ppb, such as at least 15 ppb, such as at least 20 ppb, such as at least 25 ppb, such as at least 30 ppb, such as at least 40 ppb, such as at least 50 ppb, such as at least 60 ppb, such as at least 70 ppb, such as at least 80 ppb, such as at least 90 ppb, such as of at least 100 ppb.
    [00054] In a preferred modality, the drink has an alcohol concentration of more than 0.5% (vol/vol) alcohol and no more than 1.2% (vol/vol) alcohol.
    [00055] In another preferred modality, the drink has an alcohol concentration of no more than 0.5% (vol/vol) alcohol.
    [00056] Preferably, the low-alcohol or non-alcoholic beverage is a low-alcohol or non-alcoholic beer.
    [00057] The present invention in a further aspect relates to the use of a Pichia kluyveri yeast strain for preparing a low-alcohol or non-alcoholic beverage.
    [00058] In a preferred embodiment, the low-alcohol or non-alcoholic beverage is a low-alcohol or non-alcoholic beer. EXAMPLES EXAMPLE 1 MATERIALS AND METHODS PREPARATION OF FERMENTATION
    [00059] Both low-alcohol and non-alcoholic beers were brewed with two different Pichia kluyveri strains (A strain and B strain). In total, two evaluations were carried out: 1) Non-alcoholic beer with Pichia kluyveri A strain and B strain compared to each other. 2) Low-alcohol beer with Pichia kluyveri A strain.
    [00060] The infusion formulation was initially the same for both evaluations, except for the hop addition. Non-alcoholic beer was produced on a 1500 liter scale. The low-alcohol beers with strain A and strain B from Pichia kluyveri, were produced on a 1000 liter scale. The basis for all the beers was a blend of 4 different malt types: 20% Pilsen malt, 38% Wheat malt, 38% Munich malt and 4% Cara 50 malt. The initial sugar concentration was measured to 8.3 Plato with an Anton Paar Beer Duster.
    [00061] All yeast strains were inoculated at 5x106 CFU/ml.
    [00062] For non-alcoholic beer, with a comparison between strain A and strain B of Pichia kluyveri, only hop extract was used for bittering. Fermentation was carried out at 20°C for about three weeks.
    [00063] For the low-alcohol beer, hop extract was used for bittering and at the end of cooking the wort, Tettnang and Amarillo hops were used as hop flavoring. Fermentation was carried out at 21°C. Fermentation was carried out for three weeks, and halfway through the fermentation, Tettnang and Amarillo hops were added for dry hops. The fermentation temperature was lowered to 4°C in the course of dry hopping.
    [00064] After fermentation, all beers were cooled to 4°C for several days, filtered and pasteurized. The beer was then deposited in the bottles.
    [00065] The initial glucose concentration was >1% in the first assessment, but about 0.5% in the last two assessments and the glucose concentration decreased very slowly during fermentation. At the end of fermentation, almost all glucose was consumed by the yeast and converted to ethanol.
    [00066] An overview of the infusion process is given in Figure 1, where it is compared to a normal infusion process. Important steps in the "new" brewing process are: 1) Regulate the mixing conditions to optimize the glucose concentration in the wort 2) Ensure that the added hops are sterile, then use free hop pellets or hop extracts dry hopping as this can result in contamination of the final beer. 3) A longer fermentation time to optimize the beer's flavor profile 4) Bottle pasteurization to avoid the risk of contamination due to the high level of maltose in the final beer.
    [00067] As Pichia kluyveri strains are very slow fermentation strains, it is important to keep the wort and beer sterile. Any additions of hops must be done in a sterile manner, which means at least 30 minutes before the end of the wort boil. Dry hopping (addition of hops after boiling) should not be used for the production of low-alcohol or non-alcoholic beers, as there is a risk of contamination of the beer. As a large amount of sugar is left in the final beer, special care must be taken to prevent spoilage. LABORATORY INFUSION EVALUATION
    [00068] Laboratory scale fermentation evaluations were performed on 500 ml of wort. The wort was prepared with wheat malt extract (Brewferm). The malt extract was mixed with water to achieve an initial sugar content of 10°P (Plato) as measured with an Anton Paar Beer Alcoholizer. Amarillo hop pellets were added to a closed coffee filter for the wort and the wort was boiled for 30 minutes to extract the hop flavor and to add bitterness to the beer. Hop pellets were added to reach 23 EBU (European Bitter Units). After the hop pellets had boiled, the coffee filter was removed from the wort and the wort was transferred to 1 L bottles, which were closed by water locks. Fermentation was carried out at 20°C. After 5 days of fermentation a sample was taken for diacetyl measurement. FLAVOR ANALYSIS
    [00069] Free space gas chromatography coupled with flame ionization detection (HSGC-FID) is used for the measurement of acetate esters, ethyl esters and higher alcohols in samples. Samples are analyzed with a Perkin Elmer GC System calibrated with a headspace sample. The GC is equipped with an HP-FFAP column (25 m x 0.20 mm x 0.33 µm, Agilent Technologies, Germany). The splitless split injector is used and kept at 180°C. Samples are heated for 30 minutes at 70°C in the headspace autosampler prior to injection (needle temperature: 110°C). Helium is known as the drag gas. The SOF program works as follows: after starting at 60°C, the oven temperature is raised after 2 minutes from 60°C to 230°C at 45°C/min and is finally held at 230°C for 5 minutes . During the GC program a constant flow rate (10 mL/min) of the trailing gas (He) is maintained. The FID temperature is kept constant at 220°C. Results are analyzed with Turbochrom software.
    [00070] Free space GC-MS was used for the analysis of diacetyl. In principle, this method is the same as the method used to detect flavor compounds, but an additional helium from extra split transports, which leads to an increased flow rate and results in a higher concentration of the compound in the column. Additional differences were that the pressurized bottle acts for 0.03 minutes as a drag gas reservoir and the GC program starts at a temperature of 90°C. ETHANOL AND DENSITY ANALYSIS
    [00071] Ethanol and density analysis was performed with an Anton-Paar alcoholic beer analysis system. RESULTS TABLE 2 CONCENTRATIONS (IN PPM; MG/L) OF ETHYL BUTYRATE, ISOAMYL ACETATE, ISOAMYLIC ALCOHOL, ETHYL HEXANOATE AND ETHYL OCTANOATE IN 6 COMMERCIAL BEERS AND BEER PREPARED WITH CEPA A DE PIVERICHIA AND PIVERICHIA KLUY .
    BEER WITHOUT ALCOHOL PRODUCED BY THE USE OF PICHIA KLUYVERI DURING FERMENTATION
    [00072] To produce a non-alcoholic beer, an infusion process with Pichia kluyveris yeast strain was carried out (see Materials and Methods). The Pichia kluyveri strains were used to produce a beer with an alcohol percentage of less than 0.5%. In European terms, this is considered a non-alcoholic beer. This infusion evaluation was performed on a 1000 liter scale, with Pichia kluyveri A strain and B strain and a slightly lower fermentation temperature of 20°C (see FIGURE 1 for infusion process details). All the glucose was consumed at the end of fermentation and the beer produced with strain A had an alcohol percentage of 0.1%. Beer produced with strain B had an alcohol percentage of 0.2%. Fermentation time was three weeks.
    [00073] The final two beers were used for flavor analysis. In order to get an idea of the flavor profile of non-alcoholic commercial beers, 4 non-alcoholic commercial beers were included in the flavor analysis. The 3 beers were 3 horses, Hollandia and Bavaria. The following ingredients were mentioned in the beers: - Classic non-alcoholic 3 horses beer: water, barley, malt, corn and hops. - Hollandia non-alcoholic malt beer: water, barley, malt, maltose and hop extract. - Non-alcoholic Bavarian beer: pure mineral water, barley malt, maltose and hops.
    [00074] As a control, the flavor profile of a Carlsberg lager (4.6% (vol/vol) alcohol) was used. To compare the evaluated beers infused with Pichia kluyveri with other commercial lagers, the flavor profile of two other lagers was measured: Premium Stella lager beer (5.2% (vol/vol) alcohol) and Heineken lager beer (5 ,0% (vol/vol) of alcohol).
    [00075] As can be seen from the list above, the main ingredients for non-alcoholic beers are water, malt (sometimes with unmalted barley as adjuncts) and hops. These are the same main ingredients as in reviews with Pichia kluyveri.
    [00076] The result of the flavor analysis is depicted in Figure 2. The concentrations of the 5 flavor compounds, isoamyl acetate, isoamyl alcohol, ethyl butyrate, ethyl hexanoate and ethyl octanoate are shown, each one scaled so that the contraction of the respective flavor compound for Carlsberg Lager is 1.
    [00077] Figure 2 clearly shows that all non-alcoholic commercial beers have a very low concentration of typical beer flavor compounds when compared to Carlsberg lager and compared to the other three lagers. However, beer produced with Pichia kluyveri has similar levels of beer-specific flavor compounds as the reference lager, in particular isoamyl alcohol, ethyl hexanoate and ethyl octanoate. Ethyl butyrate was present in low amounts (half the concentration) and isoamyl acetate was present in higher amounts (double or more concentration). When compared to other pilsen beers, it appears that the concentration of isoamyl acetate in Pichia kluyveri beer is even higher, as are the concentrations of ethyl octanoate. However, concentrations of ethyl butyrate and ethyl hexanoate were higher in pilsen beers than in Pichia kluyveri beer. These compounds are present in very low concentrations in beer and are less important compared to concentrations of isoamyl acetate. Isoamyl alcohol concentrations did not differ much between Pichia kluyveri and Pilsen beers, but it was virtually absent in non-alcoholic beers. When the overall flavor profile of Pichia kluyveri beer is compared to that of non-alcoholic and pilsen beers, it appears that the flavor profile of Pichia kluyveri beer is closer to a 4.6 to 5.2% beer (vol. /vol) of alcohol than the flavor profile of any of the measured non-alcoholic commercial beers.
    [00078] As these flavor compounds have a similar fruity taste, they work synergistically. Tasting the Pichia kluyveri beer it was revealed that the taste was very similar to beer and this beer was preferred over commercial non-alcoholic beers by a tasting panel consisting of infusers and beer drinkers. LOW ALCOHOLIC BEER PRODUCED WITH PICHIA KLUYVERI
    [00079] This beer was brewed by 1500 liters of wort and the fermentation temperature was 21°C (see Figure 1 for details of the brewing process). This evaluation was performed with a Pichia kluyveri A strain. All glucose was consumed at the end of fermentation and the final ethanol level was 0.7%. Fermentation time was three weeks.
    [00080] As can be seen from Figure 3, both non-alcoholic beer and low-alcohol beer prepared with the Pichia kluyveri A strain have a similar flavor profile, which means that both non-alcoholic beer and non-alcoholic beer Low-alcohol beer are very close to a pilsner beer flavor profile. Again, the main esters isoamyl acetate, ethyl butyrate, ethyl hexaonate, ethyl octanoate and isoamyl alcohol determine the flavor profile. MEASUREMENT OF DIACETYL PRODUCED BY PICHIA KLUYVERI
    [00081] To make sure that Pichia kluyveri does not produce any significant outside flavors in beer, the production of the most important outside flavor in beer, diacetyl, was measured in a laboratory infusion evaluation with Pichia kluyveri A strain alone, compared to a beer with a Saccharomyces cerevisiae strain of beer (Figure 4).
    [00082] It is clear from Figure 4 that Pichia kluyveri produces less diacetyl compared to an infusion strain of Saccharomyces cerevisiae. This is important as diacetyl is often seen as an outside flavor in beer. Especially, in non-alcoholic or low-alcohol beers where no alcohol is present, the diacetyl taste threshold level is probably much lower than in normal strong beers. CONCLUSION
    [00083] The taste profile results of alcohol-free or low-alcohol beers produced with Pichia kluyveri show that these yeast strains are ideally suited for producing beers with less or no alcohol. Especially, the production of a large amount of esters, which comes from a lower glucose concentration, is a special property seen in Pichia kluyveri strains. Along with isoamyl acetate, other esters are enhanced, which provide beers with little or no alcohol with a beer-like flavor profile. EXAMPLE 2
    [00084] Saccharomycodes ludwigii was mentioned by Branyik et al. (2012) as the most successful genus, different from Saccharomyces, used for the industrial production of non-alcoholic beer.
    [00085] To compare the properties of Pichia kluyveri with the properties of Saccharomycodes ludwigii for the preparation of low-alcohol beer, a second experiment was set up: MATERIALS AND METHODS CULTURE Yeast
    [00086] The two yeast cultures (Pichia kluyveri A strain and the Saccharomycodes ludwigii yeast strain) were prepared in YPD (2% glucose, 1% peptone and 0.5% yeast extract), with a pH adjustment of 5.5. A medium YPD was autoclaved at 121°C for 20 minutes.
    [00087] Yeast cultures were plated on YGC sheets (Sigma-Aldrich) and a single colony was inoculated into 5 ml of YPD medium. These cultures were grown overnight in a shaking incubator at 30°C and inoculated into 500 ml of YPD. After 2 days, a sample was taken by cell counts to have the ability to inoculate yeast cultures at the same starting cell count in the medium wort. LABORATORY INFUSION EVALUATION
    [00088] Laboratory scale fermentation evaluations were performed on 500 ml of must. The wort was prepared with pilsner malt extract (Brewferm). The malt extract was mixed with water to achieve an initial sugar content of 7°P (Plato) as prescribed by an Anton Paar Beer Alcoholizer. In total, 10 liters of must was prepared in this way.
    [00089] In the first assembly, the wort was boiled for 60 minutes without the addition of hops. In the second montage,
    [00090] 4.5 g Amarillo, 4.5 g Cascade and 2 g Tettnanger hop pellets were added to the wort and boiled for 60 minutes to give bitterness. 10 minutes before the end of the boil, 1.5 g Cascade, 1.5 g Amarillo and 1 g Tettnanger hop pellets were added as flavoring hops.
    [00091] After boiling, the wort was transferred to 2L bottles (1600 ml of wort per bottle), which were closed by water locks.
    [00092] Yeast cultures were inoculated at 1x106 CFU/ml and fermentation was performed at 20°C. After 5 days of fermentation, a sample was taken for flavor analysis. ETHANOL ANALYSIS
    [00093] Ethanol was measured with the Ethanol Enzymatic Bioanalysis kit from Boehringer Mannheim. FLAVOR ANALYSIS
    [00094] The flavor analysis was performed by Aroma Analysis and Enology Laboratory (Zaragoza, Spain). Smaller aroma compounds are expressed in μg/L (ppb) and are measured with solid phase extraction and Gas chromatography with mass spectrometric detection, while larger aroma compounds are expressed in mg/L (ppm) and are measured with detection of flame ionization gas chromatography after extracting the compounds and dichloromethane. RESULTS
    [00095] Laboratory scale fermentations were carried out on two types of pilsner wort: wort without the addition of hops and the same pilsner wort with the addition of bittering and aroma hops (Cascade, Amarillo and Tettnanger) (see materials and methods). Two different yeast strains were tested: Saccharomycodes ludwigii and Pichia kluyveri. Yeast cultures were inoculated at a cell count of 1E6 CFU/ml after culturing them in YPD medium. In total, 4 different fermentations were carried out: 1) Saccharomycodes ludwigii in wort without hops (S. ludwigii w/o hops) 2) Saccharomycodes ludwigii in wort with hops (S. ludwigii with hops) 3) Pichia kluyveri in wort without hops (hops) P. kluyveri w/o hops) 4) Pichia kluyveri in hop wort (P. kluyveri with hops)
    [00096] All fermentations were carried out for 5 days at 20°C.
    [00097] During fermentation, cell counts were measured every day to follow yeast growth (Figure 5).
    [00098] It is clear from Figure 5 that both yeast strains survived in the wort with and without hops. Pichia kluyveri is clearly growing compared to Saccharomycodes ludwigii, which has a stable cell count throughout fermentation.
    [00099] After 5 days, the concentration of ethanol was measured in all fermentation products (Table 3). TABLE 3: ETHANOL CONCENTRATION AND ALL 4

    [000100] Table 3 shows that both Saccharomycodes ludwigii and Pichia kluyveri produce very small amounts of ethanol. Pichia kluyveri seems better at producing no alcohol as its strain grows to higher cell counts, but produces less alcohol compared to Saccharomycodes ludwigii.
    [000101] Fermentation products on day 5 were also analyzed for the flavor profile (Figures 6, 7, 8 and 9). The most important flavor compounds for beer are the higher esters and alcohols.
    [000102] Figures 6 and 7 show the ester concentrations in all final fermentation products, as well as in the basic wort with and without hops. All ester concentrations are highest in Pichia kluyveri fermentation products, either with or without hops. In the case of isoamyl, phenylethyl, isobutyl and butyl acetates, Pichia kluyveri produces higher amounts of these esters when no hops are added, purchased from ethyl decanoate. Especially the levels of isoamyl acetate and phenylethyl acetate are quite high. Isoamyl acetate results in a banana aroma, while phenylethyl acetate results in a more floral, honey aroma. Both ester compounds are desirable in beer. It should be noted that the results described here are ester concentrations after 5 days of fermentation, and these concentrations decrease over time in a final beer. It is important to have high concentrations of ester to start, which results in a fruity beer for a longer period of time as these ester compounds are hydrolyzed over time into a final beer.
    [000103] The concentrations of fusel alcohol, described in FIGURE 8, are quite similar between the fermentation products of Saccharomycodes ludwigii and Pichia kluyveri. Only isoamyl alcohol concentrations are higher in Saccharomycodes ludwigii fermentation products, probably because this compound is not converted to isoamyl acetate, as is the case in Pichia kluyveri fermentation products.
    [000104] In Figure 9, three acids are represented, which are negative flavor compounds in beer. Especially decanoic acid which gives a stale, cheesy taste to beer and is therefore undesirable. It is very clear that especially Saccharomycodes ludwigii is produced with large amounts of these acids, which makes it less suitable for brewing. CONCLUSION
    [000105] Laboratory infusion evaluation with the two different non-Saccharomyces yeast strains clearly indicate that Pichia kluyveri is better suited for the production of non-alcoholic beer compared to Saccharomycodes ludwigii. Pichia kluyveri produces less alcohol, more desired ester compounds and less unwanted acids compared to Saccharomycodes ludwigii. DEPOSITS
    [000106] Pichia kluyveri strains PK-KR1 (JT1.28 or strain A) and PK-KR2 (JT3.71) have been deposited on August 24, 2006 at the National Measurement Institute, 51 to 65 Clarke Street, South Melbourne , Victoria 3205, Australia, by the University of Auckland, School of Biological Sciences, Auckland 1142, New Zealand, and given accession numbers V06/022711 and V06/022712 as described in WO 2009/110807. REFERENCES DD 288619 WO 2009; 110807. Boulton, C.M. and Quain, D. 2010. Brewing yeast and fermentation. Blackwell publishing Ltd. Branyik, T, Silva, D.P., Baszczyn, M., Lehnert, R., Almeida and Silva, J.B., 2012. A review of methods of low alcohol and alcohol-free beer production. Journal of Food Engineering 108,493-506. Liu, Y., Li, H., Du, J.-H. 2011. Non-alcoholic Beer Production by Saccharomycodes ludwigii. Food Science, 32: 186 to 190. Montanari, L., Marconi, O., Mayer, H., Fantozzi, P., 2009. Production of alcohol-free beer. In: Preedy, V.R. (Ed.), Beer in Health and Disease Prevention. Elsevier Inc., Burlington, MA, 61-75. Saerens S.M., Delvaux F.R., Verstrepen K.J., Thevelein J.M. 2010. Production and biological function of volatile esters in Saccharomyces cerevisiae. Microbial Biotechnology 3:165 to 77. Stewart, G. Wort glucose, maltose or maltotriose - from brewer's yeast strains care which one Institute of Brewing and Distilling. Asia Pacific Section. 31st Asia Pacific Convention 2010. (http://www.ibdasiapac.com.au/asia-pacific-activities/convention-proceedings/2010/results.html) Swiegers, J.H., S.M.G. Saerens, I.S. Praetorian. 2007. The development of yeast strains as tools to adjust the flavor of fermented beverage to market specifications. Havkin D. and Belanger F. Biotechnology in flavor production. Blackwell publishing Ltd. Zufall C, and Wackerbauer K. 2000. Process engineering parameters for the dealcoholization of beer by means of falling film evaporation and its influence on beer quality. Monatsschrift für Brauwissenschaft 53:124 to 137.
    权利要求:
    Claims (6)
    [0001]
    1. Method of preparing a low-alcohol or non-alcoholic beverage, with an alcohol content of not more than 1.2% (vol/vol) and an isoamyl acetate content of 0.5 ppm or more, characterized by steps of: a) providing a wort; b) add a variety of hops to the wort; and c) fermenting the wort with a Pichia kluyveri yeast strain to obtain the low-alcohol or alcohol-free beverage under sterile conditions and with Pichia kluyveri as the only added yeast strain.
    [0002]
    2. Method according to claim 1, characterized in that the fermentation of the must with the Pichia kluyveri yeast strain is carried out for 7 days.
    [0003]
    3. Method according to claim 1 or 2, characterized in that the fermentation of the must with the Pichia kluyveri yeast strain is carried out at a temperature of between 18 and 22°C.
    [0004]
    4. Method according to any one of claims 1 to 3, characterized in that the Pichia kluyveri yeast strain has the ability to ferment glucose, but not maltose or maltotriose.
    [0005]
    5. Method according to any one of claims 1 to 4, characterized in that the Pichia kluyveri strain is selected from the group consisting of Pichia kluyveri PK-KR1 strain (JT1.28 or A strain), which was deposited August 24, 2006 at the National Institute of Measurement, 541-65 Clarke Street, South Melbourne, Victoria 3205, Australia, by the University of Auckland, School of Biological Sciences, Auckland 1142, New Zealand, under accession numbers V06/022711 , and Pichia kluyveri PK-KR2 (JT3.71), which was deposited on August 24, 2006 at the National Institute of Measurement, 541-65 Clarke Street, South Melbourne, Victoria 3205, Australia, by the University of Auckland, School of Biological Sciences, Auckland 1142, New Zealand, through accession numbers V06/022712.
    [0006]
    6. Method according to any one of claims 1 to 5, characterized by the fact that the low-alcohol or non-alcoholic beverage is a beer.
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    同族专利:
    公开号 | 公开日
    BR112015021810A2|2017-07-18|
    US20160010042A1|2016-01-14|
    US20200080034A1|2020-03-12|
    ES2659745T3|2018-03-19|
    AU2014224578A1|2015-09-24|
    EP2964742A2|2016-01-13|
    US9580675B2|2017-02-28|
    US20170226451A1|2017-08-10|
    AR115812A2|2021-03-03|
    AU2014224578B2|2017-03-16|
    AP2015008705A0|2015-09-30|
    ES2800078T3|2020-12-23|
    DK2964742T3|2018-01-29|
    PE20151614A1|2015-12-03|
    WO2014135673A2|2014-09-12|
    US10415007B2|2019-09-17|
    ZA201506346B|2017-03-29|
    US11162059B2|2021-11-02|
    HK1212725A1|2016-06-17|
    DK3354717T3|2020-05-11|
    HUE036194T2|2018-06-28|
    CN105121619B|2018-03-27|
    CN109161450A|2019-01-08|
    EP3354717A1|2018-08-01|
    CN105121619A|2015-12-02|
    EP3354717B1|2020-02-26|
    EP2964742B1|2017-12-13|
    PL2964742T3|2018-05-30|
    HUE049106T2|2020-09-28|
    PE20191358A1|2019-10-01|
    AR095050A1|2015-09-16|
    WO2014135673A3|2014-10-30|
    LT3354717T|2020-06-10|
    PL3354717T3|2020-08-24|
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    法律状态:
    2018-02-27| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|
    2019-08-20| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|
    2020-11-03| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|
    2021-04-06| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|
    2021-06-22| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 07/03/2014, OBSERVADAS AS CONDICOES LEGAIS. |
    优先权:
    申请号 | 申请日 | 专利标题
    EP13158261|2013-03-07|
    EP13158261.1|2013-03-07|
    PCT/EP2014/054415|WO2014135673A2|2013-03-07|2014-03-07|Production of low-alcohol or alcohol-free beer with pichia kluyveri yeast strains|
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