OA17477A - Production of low-alcohol or alcohol-free beer with pichia kluyveri yeast strains. - Google Patents
Production of low-alcohol or alcohol-free beer with pichia kluyveri yeast strains. Download PDFInfo
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- OA17477A OA17477A OA1201500360 OA17477A OA 17477 A OA17477 A OA 17477A OA 1201500360 OA1201500360 OA 1201500360 OA 17477 A OA17477 A OA 17477A
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- Prior art keywords
- alcohol
- beer
- pichia kluyveri
- wort
- low
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- 235000013405 beer Nutrition 0.000 title claims abstract description 166
- 241001489192 Pichia kluyveri Species 0.000 title claims abstract description 93
- 240000004808 Saccharomyces cerevisiae Species 0.000 title claims abstract description 65
- 238000004519 manufacturing process Methods 0.000 title description 11
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 92
- 238000000855 fermentation Methods 0.000 claims abstract description 63
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- 235000019520 non-alcoholic beverage Nutrition 0.000 claims abstract description 28
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- 239000008103 glucose Substances 0.000 claims abstract description 22
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- 235000009808 lpulo Nutrition 0.000 claims description 15
- 238000005259 measurement Methods 0.000 claims description 8
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- 239000000796 flavoring agent Substances 0.000 abstract description 68
- 235000019634 flavors Nutrition 0.000 abstract description 61
- 150000001875 compounds Chemical class 0.000 abstract description 32
- 229940117955 isoamyl acetate Drugs 0.000 abstract description 18
- MHPUGCYGQWGLJL-UHFFFAOYSA-M 5-methylhexanoate Chemical compound CC(C)CCCC([O-])=O MHPUGCYGQWGLJL-UHFFFAOYSA-M 0.000 abstract description 17
- PHTQWCKDNZKARW-UHFFFAOYSA-N Isoamyl alcohol Chemical compound CC(C)CCO PHTQWCKDNZKARW-UHFFFAOYSA-N 0.000 abstract description 11
- YYZUSRORWSJGET-UHFFFAOYSA-N ethyl octanoate Chemical compound CCCCCCCC(=O)OCC YYZUSRORWSJGET-UHFFFAOYSA-N 0.000 abstract description 11
- OBNCKNCVKJNDBV-UHFFFAOYSA-N Ethyl butyrate Chemical compound CCCC(=O)OCC OBNCKNCVKJNDBV-UHFFFAOYSA-N 0.000 abstract description 8
- SHZIWNPUGXLXDT-UHFFFAOYSA-N ethyl hexanoate Chemical compound CCCCCC(=O)OCC SHZIWNPUGXLXDT-UHFFFAOYSA-N 0.000 abstract description 8
- 241000235070 Saccharomyces Species 0.000 abstract description 5
- 239000000758 substrate Substances 0.000 abstract description 2
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- 241001489222 Saccharomycodes ludwigii Species 0.000 description 19
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- 238000000034 method Methods 0.000 description 18
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- 235000003534 Saccharomyces carlsbergensis Nutrition 0.000 description 7
- 241001123227 Saccharomyces pastorianus Species 0.000 description 6
- 240000005979 Hordeum vulgare Species 0.000 description 5
- 235000007340 Hordeum vulgare Nutrition 0.000 description 5
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- -1 compounds isoamyl acetate Chemical class 0.000 description 5
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- GHVNFZFCNZKVNT-UHFFFAOYSA-N Decanoic acid Chemical compound CCCCCCCCCC(O)=O GHVNFZFCNZKVNT-UHFFFAOYSA-N 0.000 description 4
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- 150000001298 alcohols Chemical class 0.000 description 4
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- MDHYEMXUFSJLGV-UHFFFAOYSA-N phenethyl acetate Chemical compound CC(=O)OCCC1=CC=CC=C1 MDHYEMXUFSJLGV-UHFFFAOYSA-N 0.000 description 4
- 150000008163 sugars Chemical class 0.000 description 4
- 241000235648 Pichia Species 0.000 description 3
- 241000862969 Stella Species 0.000 description 3
- 240000008529 Triticum aestivum Species 0.000 description 3
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- 240000005616 Vigna mungo var. mungo Species 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 150000007513 acids Chemical class 0.000 description 3
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- 235000021307 wheat Nutrition 0.000 description 3
- 239000007222 ypd medium Substances 0.000 description 3
- WWZKQHOCKIZLMA-UHFFFAOYSA-N Caprylic acid Chemical compound CCCCCCCC(O)=O WWZKQHOCKIZLMA-UHFFFAOYSA-N 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- BJHIKXHVCXFQLS-UYFOZJQFSA-N Fructose Natural products OC[C@@H](O)[C@@H](O)[C@H](O)C(=O)CO BJHIKXHVCXFQLS-UYFOZJQFSA-N 0.000 description 2
- FUZZWVXGSFPDMH-UHFFFAOYSA-N Hexanoic acid Chemical compound CCCCCC(O)=O FUZZWVXGSFPDMH-UHFFFAOYSA-N 0.000 description 2
- ZXEKIIBDNHEJCQ-UHFFFAOYSA-N Isobutanol Chemical compound CC(C)CO ZXEKIIBDNHEJCQ-UHFFFAOYSA-N 0.000 description 2
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- 235000020016 small beer Nutrition 0.000 description 2
- 241000722885 Brettanomyces Species 0.000 description 1
- ZUEMSFSFDAWANS-UHFFFAOYSA-N CCCC(=O)OCC.CC(C)CCOC(C)=O Chemical compound CCCC(=O)OCC.CC(C)CCOC(C)=O ZUEMSFSFDAWANS-UHFFFAOYSA-N 0.000 description 1
- 229940041514 Candida albicans extract Drugs 0.000 description 1
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- 240000006245 Dichrostachys cinerea Species 0.000 description 1
- RGXWDWUGBIJHDO-UHFFFAOYSA-N Ethyl decanoate Chemical compound CCCCCCCCCC(=O)OCC RGXWDWUGBIJHDO-UHFFFAOYSA-N 0.000 description 1
- 239000005715 Fructose Substances 0.000 description 1
- 241000235042 Millerozyma farinosa Species 0.000 description 1
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- CZMRCDWAGMRECN-GDQSFJPYSA-N Sucrose Natural products O([C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](CO)O1)[C@@]1(CO)[C@H](O)[C@@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-GDQSFJPYSA-N 0.000 description 1
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- 235000016383 Zea mays subsp huehuetenangensis Nutrition 0.000 description 1
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Abstract
It has unexpectedly been found that a low alcohol or alcohol-free beverage, with a flavor profile very close to a beer of at least 4% (vol/vol) alcohol, can be produced by using Pichia kluyveri yeast strains. In particular, Pichia kluyveri yeast strains only use the glucose in the wort, and have the ability of converting this substrate into a high concentration of specific flavor compounds, which are normally produced by Saccharomyces ssp. yeast strains used for the brewing of beer. In this way the Pichia kluyveri yeast strains can be used to produce either a low alcohol or alcohol-free beverage, depending on the glucose levels in the wort. The main flavor compounds produced by Pichia kluyveri in the fermentation of wort are isoamyl acetate, isoamyl alcohol, ethyl butyrate, ethyl hexanoate and ethyl octanoate.
Description
ORIGINAL
DEMANDE DE BREVETD’ INVENTION
Chr. Hansen A/S
Production of low-alcohol or alcohol-free beer with pichia kluyveri yeast strains
INVENTEURS : SAERENS Sofie
M
SWIEGERS Jan Hendrik
Chr. Hansen A/S
PRODUCTION OF LOW-ALCOHOL OR ALCOHOL-FREE BEER WITH
PICHIA KLUYVERI YEAST STRAINS
FIELD OF THE INVENTION
The présent 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 alcohol-free beverage with enhanced levels of désirable fermentationderived flavor compounds comprising a step of fermentation of a wort with a Pichia kluyveri yeast strain to obtain a low-alcohol or alcohol-free beverage, a low-alcohol or alcohol-free beverage obtainable by the method and a low-alcohol or alcohol-free beverage comprising a high concentration of spécifie flavor compounds, such as isoamyl acetate and ethyl octanoate.
BACKGROUND OF THE INVENTION
Due to the increasing demand for healthier food and beverages, the réduction of éthanol in alcoholic beverages, especially beer and wine, is of considérable commercial interest.
Low-alcohol, alcohol-free or reduced-alcohol beers (also called light beer, no-alcohol, non-alcoholic beer, small beer, small ale, near-beer) are beers with no alcohol or a low alcohol content which aim to reproduce the full flavor of standard beers which normally contain more than 4% (vol/vol) alcohol. In most of the EU countries beers with low alcohol content are divided into alcohol-free beers containing no more than 0.5% (vol/vol) alcohol, and into low-alcohol beers with no more than l.2% (vol/vol) alcohol. In the United States alcohol-free beer means that there is no alcohol présent, while the upper limit of 0.5% (vol/vol) alcohol corresponds to so-called non-alcoholic or near-beer (Montanari et al. 2009).
The conversion of wort into an alcoholic beer is the resuit of a fermentation process by
Saccharomyces ssp. yeast resulting in the production of alcohol and fermentation-derived flavor compounds, such as esters (e.g. isoamyl acetate) and higher alcohols. Isoamyl — t
acetate is a key flavor compound in beer (Saerens et al. 2010). The combination of the flavor of the raw materials, mostly 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).
Today beers with low alcohol contents are often made by producing full-strength beer (with an alcohol concentration of above 4% (vol/vol)) and then removing the alcohol by physical processes (boiling off the alcohol/distillation or reverse osmosis). In the case of some non-alcoholic or alcohol-free beers no fermentation takes place and in some cases the beers are produced by simply diluting full-strength beers with water.
As the physical processes resuit in the évaporation, loss or dilution of fermentationderived flavor compounds (or a complété lack of fermentation-derived flavor compounds as in the case with unfermented non-alcoholic or alcohol-free beer), these types of beers are often characterized as being less flavorful than full-strength beer or may hâve an undesirable wort-like taste (Zufall and Wackerbauer. 2000).
German patent DD 288619 A5 describes the use of Pichia farinosa yeast strains to ferment unhopped beer wort to obtain an alcohol-free beverage with fruity aroma.
However, there exists a need for improved methods for preparing a low-alcohol or alcohol-free fermented beverage wherein the presence of désirable flavor compounds, such as esters and higher alcohols, is enhanced.
SUMMARY OF THE INVENTION
The présent invention is based on the surprising findings by the inventors that Pichia kluyveri yeast strains, only capable of fermenting the glucose in beer wort, is capable of producing high concentrations of désirable flavor compounds with only little éthanol production.
The brewing yeasts of Saccharomyces pastorianus, Saccharomyces carlsbergensis and
Saccharomyces cerevisiae utilize both glucose as well as maltose, the main sugars in wort (Boulton and Quain. 2010) resulting 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. In order to produce less alcohol, a solution is to use a yeast that only ferments glucose and not maltose, thereby only producing alcohol from the glucose. Glucose is présent in much lower concentrations than maltose in wort (Table 1) and therefore less alcohol will be produced from the same amount of wort compared to when a conventional brewing yeast, e.g. Saccharomycespastorianus or Saccharomyces carlsbergensis, is used. These types of yeasts exist and examples include certain Saccharomyces and non-Saccharomyces yeast strains, such as Saccharomycodes ssp (Liu étal. 2011).
However, only a relatively small amount of flavor will be produced by these yeast strains from the limited amount of glucose resulting in a beer with lower concentrations of key flavor compounds than beers with more than 4% (vol/vol) alcohol.
Table 1. Typical sugar spectrum of wort (Stewart. 2010
| Percent composition | |
| Glucose | 10-15 |
| Fructose | 1 -2 |
| Sucrose | 1 -2 |
| Maltose | 50-60 |
| Maltotriose | 15-20 |
| Dextrins | 20-30 |
One way of solving this problem, is to identify a highly aromatic yeast, which is not able to utilize the maltose, in order to produce high concentrations of relevant fermentationderived flavor compounds from limited amounts of glucose, resulting in similar or higher concentrations of key flavor compounds than when the full fermentation takes place. The inventors of the présent invention hâve found that by using this method and Pichia kluyveri yeast strains, it is possible to produce alcohol-free beer and low-alcohol beer (according to EU législation) with the same flavor as standard, full-strength beers. This is not possible using conventional brewing yeast such as Saccharomyces cerevisiae and Saccharomyces pastorianus or Saccharomyces carlsbergensis, dilution, physical alcohol removal processes or beers produced form non-fermented substrates.
Accordingly, a first aspect of the invention relates to a method of preparing a low-alcohol or alcohol-free beverage comprising the steps of:
a) providing a wort; and
b) fermenting the wort with at least one Pichia kluyveri yeast strain to obtain a low-alcohol or alcohol-free beverage.
An additional aspect of the présent invention relates to a low-alcohol or alcohol-free beverage obtainable by the method according to the first aspect of the invention.
A further aspect of the présent invention relates to a low-alcohol or alcohol-free beverage, wherein the low-alcohol or alcohol-free beverage has an isoamyl acetate content of at least 0.5 ppm (mg/L).
An even further aspect of the présent invention relates to use of a Pichia kluyveri yeast strain for preparing low-alcohol or alcohol-free beverage.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGURE 1 illustrâtes an optimized beer brewing process for Pichia kluyveri beer. Comparison of the standard (left) versus Pichia kluyveri (right) brewing process. Steps that are different from the standard brewing process are highlighted.
FIGURE 2 illustrâtes the flavor profile comparison of 5 different alcohol-free beers: 3 horses. Hollandia, Bavaria (ail with 0% alcohol) and the beers produced with Pichia kluyveri strain A (0.1% (vol/vol) alcohol) and strain B (0.2% (vol/vol) alcohol). Carlsberg pilsner beer with 4.6 % (vol/vol) alcohol was used as a reference pilsner beer and two other pilsner beers are shown as well: 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 pilsner beer were used as a reference and were scaled to be 1. The flavor concentrations in ail beers were put on the same scale in order to be able to compare the values.
FIGURE 3 shows flavor profile comparison of 4 different beers: alcohol-ffee beer with Pichia kluyveri strain A (0.1% (vol/vol) alcohol), low alcohol beer with Pichia kluyveri strain A (0.7% (vol/vol) alcohol), Carlsberg pilsner beer (4.6% (vol/vol) alcohol) which was used as a reference pilsner beer (control) and malt beer (1.8% (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 pilsner beer were used as a reference and were scaled to be 1. The flavor concentrations in the 3 beers were put on the same scale in order to be able to compare the values.
FIGURE 4 illustrâtes the diacetyl concentrations in beer trials with Pichia kluyveri strain A and a Saccharomyces cerevisiae beer yeast.
FIGURE 5 depicts yeast cell growth during fermentation.
FIGURE 6 shows isoamyl acetate ethyl decanoate and phenylethyl acetate concentrations in ail fermentation products with Saccharomycodes ludwigii and Pichia kluyveri, as wll as in the basic wort (with and without hops). w/o = without.
Figure 7 illustrâtes isobutyl acetate and butyl acetate concentrations in ail fermentation products with Saccharomycodes ludwigii and Pichia kluyveri, as well as in the basic wort (with and without hops). w/o = without.
FIGURE 8 shows isobutanol, 1-butanol and isoamyl alcohol concentrations in ail fermentation products with Saccharomycodes ludwigii and Pichia kluyveri, as well as in the basic wort (with and without hops). w/o = without.
FIGURE 9 depicts hexanoic, octanoic and decanoic acid concentrations in ail fermentation products with Saccharomycodes ludwigii and Pichia kluyveri, as well as in the basic wort (with and without hops). w/o = without. Y
DETAILED DESCRIPTION OF THE INVENTION
The inventors of the présent invention hâve unexpectedly found that Pichia kluyveri yeast strains are able to enhance the presence of désirable flavor compounds in beverages prepared by fermenting wort with the Pichia kluyveri yeast strains. In particular, it was found that concentrations of flavor compounds désirable for beer, such as isoamyl acetate, isoamyl alcohol and ethyl octanoate, were increased.
Surprisingly, this enhancement of beer flavor compounds is présent when yeast strains of the species Pichia kluyveri are used in the absence of conventional brewing yeast strains, such as yeast strains of the généra Saccharomyces and Brettanomyces.
The process of brewing beer is well known to the skilled person and can be outlined in the following way (see Figure l); malt is prepared from dried, germinated cereal grains (mainly barley or wheat) and grounded into a grist which may contain unmalted adjuncts. The grist is mashed (mixed with water and steeped) to allow 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 lautering. The malt making and mashing steps can be skipped by adding water to malt extract. After addition of hops and/or other ingrédients such as herbs and sugars, the wort is boiled (hops may also be added after boiling), cooled and aerated. The wort is then moved to a fermentation tank and fermented by the addition of a brewer’s yeast. The primary fermentation, lasting typically 5 to 10 days, may be foliowed by a secondary fermentation step using a brewer’s yeast. After fermentation the fresh beer or “green” beer, is conditioned, optionally filtrated and carbonated. The beer is filled on bottles and optionally pasteurized.
Hops can be added to the wort to balance the sweetness of the malt with bittemess and impart onto the beer désirable flavors and aromas. Several varieties exist including but not limited to Ahtanum, Amarillo, Apollo, Bravo, Calypso, Cascade, Centennial, Chelan, Chinook, Citra, Cluster, Columbus, Cornet, 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, Admirai, Brewer’s Gold, Bramling Cross, Bullion, Challenger, First Gold, Fuggles, —
Goldings, Herald, Northdown, Northem Brewer, Phoenix, Pilgrim, Pilot, Pioneer, Progress, Target, Whitbread Golding Variety (WGV), Hallertau, Hersbrucker, Saaz, Tettnang, Spalt, Ella, Feux-Coeur Français, Galaxy, Green Bullet, Hallertau Aroma, Kohatu, Motueka, Nelson Sauvin, Pacifie Gem, Pacifie Jade, Pacifica, Pride of Ringwood, Rakau, Riwaka, Southem 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.
The method for preparing a low-alcohol or alcohol-free beverage of the présent invention comprises the steps of:
a) providing a wort; and
b) fermenting the wort with at least one Pichia kluyveri yeast strain to obtain a low-alcohol or alcohol-free beverage.
The term “low-alcohol beverage” herein is defîned as a liquid for drinking with an alcohol content of more than 0.5% (vol/vol) of alcohol and no more than 1.2% (vol/vol) of alcohol.
The term “alcohol-free beverage” herein is defîned as a liquid for drinking with an alcohol content of no more than 0.5% (vol/vol) of alcohol.
The term “wort” herein has the conventional meaning in the art and refers to the sugary liquid extracted from the mashing process of beer brewing.
The concentration of glucose in the wort is critical for the resulting alcohol percentage of the beverage and can be adjusted by changing the mashing process, by addition of enzymes and by other methods known to the skilled person.
In a preferred embodiment the wort has a concentration of glucose between 0.01 % to 3% (w/vol), such as between 0.5% to 1.0% (w/vol).
Preferably, the fermentation in step b) is carried out under stérile conditions (controlled fermentation) and with Pichia kluyveri as the only species of yeast strain added.
The skilled person will readily be able to détermine a concentration of inoculation of the Pichia kluyveri yeast strain suitable for préparation of a low-alcohol or alcohol-free beverage according to the method.
In a preferred embodiment, the Pichia kluyveri yeast strain is inoculated in a concentration of at least lxlO4 CFU/ml (colony-forming units/ml), such as at least 5x104 CFU/ml, such as at least lxlO5 CFU/ml, such as at least 5x105 CFU/ml, such as at least lxlO6 CFU/ml, such as at least 5x106 CFU/ml.
In another preferred embodiment the method further comprises the step al) adding to the wort at least one hop variety prior to fermenting the wort with the at least one Pichia kluyveri yeast strain.
In another preferred embodiment the fermentation of the wort with the 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 28 days.
In another preferred embodiment the fermentation of the wort with the at least one Pichia kluyveri yeast strain is carried out at a température of between 18°C and 22°C.
In yet another preferred embodiment the method further comprises the step c) bottling and bottle pasteurizing the low-alcohol or alcohol-free beverage.
Preferably, the at least one Pichia kluyveri yeast strain is capable of fermenting glucose but not maltose or maltotriose. This can be tested by growing the yeast strain on a culture medium with only maltose or maltotriose.
In a preferred embodiment the at least one Pichia kluyveri yeast strain is selected from the group consisting of the Pichia kluyveri PK-KR1 (JT1.28; strain A), that was deposited on
August 2006 at the National Measurement Institute, 541-65 Clarke Street, South
Melbourne, Victoria 3205, Australia, by University of Auckland, School of Biological
Sciences, Auckland 1142, New Zealand, under accession numbers V06/022711, and the
Pichia kluyveri PK-KR2 (JT3.71), that was deposited on 24 August 2006 at the National
Measurement Institute, 541-65 Clarke Street, South Melbourne, Victoria 3205, Australia, by University of Auckland, School of Biological Sciences, Auckland 1142, New Zealand, under accession numbers V06/022712.
Preferably the low-alcohol or alcohol-free beverage is a low-alcohol or alcohol-free beer.
The term “beer” as used herein refers at least to beers prepared from mashes prepared from malted cereals as well as mashes prepared from unmalted cereals, and mashes prepared from a mixture of malted and unmalted cereals. The term “beer” also refers to beers prepared with adjuncts and beers with ail possible alcohol contents.
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) of alcohol.
The term “non-alcohol beer” herein refers to a beer with an alcohol content of no more than 0.5% ABV.
An additional aspect of the présent invention relates to a low-alcohol or alcohol-free beverage obtainable by the method described herein.
In a preferred embodiment the low-alcohol or alcohol-free beverage is a low-alcohol or alcohol-free beer.
A further aspect of the présent invention is directed to a low-alcohol or alcohol-free 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.
ίο
Preferably, the low-alcohol or alcohol-ffee beverage has an ethyl octanoate concentration of at least 10 ppb (parts per billion; pg/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 at least 100 ppb.
In a preferred embodiment the beverage has an alcohol concentration of more than 0.5% (vol/vol) alcohol and no more than 1.2% (vol/vol) alcohol.
In another preferred embodiment the beverage has an alcohol concentration of no more than 0.5% (vol/vol) alcohol.
Preferably, the low-alcohol or alcohol-free beverage is a low-alcohol or alcohol-ffee beer.
The présent invention in one further aspect relates to use of a Pichia kluyveri yeast strain for preparing a low-alcohol or alcohol-free beverage.
In a preferred embodiment the low-alcohol or alcohol-free beverage is a low-alcohol or alcohol-free beer.
EXAMPLES
Example 1
Materials and methods
Fermentation set-up
Both low alcohol and alcohol-free beer were produced with 2 different Pichia kluyveri strains (strain A and strain B). In total 2 trials hâve been carried out:
1) Alcohol-free beer with Pichia kluyveri strain A and strain B compared to each other.
2) Low alcohol beer with Pichia kluyveri, strain A.
The brewing recipe was initially the same for both trials, except for the hop addition. The alcohol-free beer was produced on a 1500 liter scale. The low alcohol beers with Pichia kluyveri, strain A and strain B, were produced on a 1000 liter scale. The basis for ail the beers was a mixture of 4 different kinds of malt: pilsner malt 20%, wheat malt 38%, Munich malt 38% and Cara 50 malt 4%. The initial sugar concentration was measured to 8.3°Plato with an Anton Paar Beer Alcolyzer.
Ail yeast strains were inoculated at 5x106 CFU/ml.
For the alcohol-free beer, with a comparison between strain A and strain B of Pichia kluyveri, only hop extract was used for the bittemess. The fermentation was carried out at 20°C for about three weeks.
For the low alcohol beer, hop extract was used for the bittemess and at the end of wort cooking, Tettnang and Amarillo hops were used as aroma hops. The fermentation was carried out at 21°C. The fermentation was carried out for three weeks, and in the middle of fermentation, Tettnang and Amarillo hops were added as hop bells for dry hopping. The fermentation température was lowered to 4°C in the course of the dry hopping.
After fermentation, ail beers were cooled down to 4°C for several days, filtered and pasteurized. The beer was then filled in bottles.
The initial glucose concentration was >1% in the first trial, but around 0.5% in the last two trials and the glucose concentration decreased very slowly during fermentation. At the end of fermentation, almost ail glucose was consumed by the yeast and converted to éthanol.
An overview of the brewing process is given in Figure 1, where it is compared to a normal brewing process. Important steps in the ‘new’ brewing process are:
1) Regulating the mashing conditions to optimize the glucose concentration in the wort
2) Assure that the hop added is stérile, so using hop pellets or hop extracts, no dry hopping, as this can resuit in contamination of the final beer
3) A longer fermentation time to optimize the flavor profile of the beer
4) Bottle pasteurization to avoid the risk of contamination due to the high maltose level in the final beer. -νχΥ
As Pichia kluyveri strains are very slow fermenting strains, it is important to keep the wort and the beer stérile. Any addition of hops should be made in a stérile manner, meaning at least 30 min. before the end of boiling of the wort. Dry hopping (addition of hops after boiling) should not be used for the production of low-alcohol or alcohol-ffee beers, as there is a risk of contaminating the beer. As a lot of sugar is left in the final beer, extra care should be taken to avoid spoilage.
Lab brewing trial
Lab-scale fermentation trials were carried out in 500 ml of wort. The wort was prepared with wheat malt extract (Brewferm). The malt extract was mixed with water to reach an initial sugar content of 10°P (Plato) as measured with an Anton Paar Beer Alcolyzer. Amarillo hop pellets were added in a closed coffee filter to the wort and this was boiled for 30 min. to extract the hop flavor and to add bittemess to the beer. Hop pellets were added to reach 23 EBU (European Bittemess Units). After boiling the, the coffee filter was removed from the wort and the wort was transferred to 1L 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
Headspace gas chromatography coupled with flame ionization détection (HSGC-FID) is used for the measurement of acetate esters, ethyl esters and higher alcohols in the samples. Samples are analyzed with a calibrated Perkin Elmer GC System with a headspace sampler. The GC is equipped with a HP-FFAP column (25 m x 0.20 mm x 0.33 pm, Agilent Technologies, Germany). The split-splitless injector is used and held at 180°C. Samples are heated for 30 min. at 70°C in the headspace autosampler before injection (needle température: 110°C). Hélium is used as the carrier gas. The SOF-program works as follows: after starting at 60°C, the oven température is raised after 2 min. from 60°C to 230°C at 45°C/min and is finally held at 230°C for 5 min. During the GC-program a constant flow rate (10 mL/min) of the carrier gas (He) is maintained. The FID température is kept constant at 220°C. The results are analyzed with Turbochrom software. —
I3
Headspace GC-MS was used for the analysis of diacetyl. In principal, this method is the same as the method used to detect flavor compounds, but an extra split transports additional hélium, leading to an increased flow rate and resulting in a higher concentration of the compound on the column. Further différences were that the pressurized vial acts for
0.03 min. as a carrier gas réservoir and the GC program starts at a température of 90°C.
Ethanol and density analysis
Ethanol and density analysis was performed with an alcolyzer beer analyzing system from Anton-Paar.
Results
Table 2: Concentrations (in ppm; mg/L) of ethyl butyrate, isoamyl acetate, isoamyl alcohol, ethyl hexanoate and ethyl octanoate in 6 commercial beers and beer prepared with 15 Pichia kluyveri strain A and Pichia kluyveri strain B.
| Ethyl butyrate | Isoamyl acetate | Isoamyl alcohol | Ethyl hexanoate | Ethyl octanoate | |
| Carlsberg | ϊ,ϊό | ' i,o‘o ' | 34,0 | 0,04 | 0,10 ” |
| Stella | 2,14 | 0,71 | 50,0 | 0,11 | 0,10 |
| Heineken | 2,02 | 4,87 | 46,0 | 0,10 | 0,09 |
| 3 horses | 0,00 | 0,00 | 0,0 | 0,00 | 0,00 |
| Hollandia | 0,00 | 0,26 | 3,2 | 0,00 | 0,00 |
| Bavaria | 0,00 | 0,18 | 2,0 | 0,00 | 0,00 |
| Malt beer | 0,71 | 0,21 | 11 | 0,01 | 0,01 |
| Pichia A - | |||||
| 0,43 | 1,96 | 2,00 | 0,03 | 0,12 | |
| alcohol-free | |||||
| Pichia B - | |||||
| 0,26 | 4,94 | 2,00 | 0,07 | 0,13 | |
| alcohol-free | |||||
| Pichia A - | |||||
| 0,27 | 2,50 | 1,80 | 0,02 | 0,13 | |
| low alcohol |
Alcohol-free beer produced by the use ο/Pichia kluyveri during fermentation
To produce an alcohol-free beer, a brewing process with Pichia kluyveri yeast strains was carried out (see Materials and methods). The Pichia kluyveri strains were used to produce a beer with an alcohol percentage less than 0.5%. In European terms, this is considered a non-alcoholic beer. This brewing trial was performed in a 1000 liter scale, with Pichia kluyveri strain A and strain B and a slightly lower fermentation température of 20°C (see Figure 1 for brewing process details). Ail the glucose was consumed at the end of fermentation and the beer produced with strain A had an alcohol percentage of 0.1%. The beer produced with strain B had an alcohol percentage of 0.2%. The fermentation time was three weeks.
The two final beers were used for flavor analysis. In order to hâve an idea of the flavor profile of commercial alcohol-free beers, 4 commercial alcohol-free beers were included in the flavor analysis. The 3 beers were 3 horses, Hollandia and Bavaria. The following ingrédients were mentioned on the beers:
- 3 horses classic non-alcoholic beer: water, barley, malt, maize and hops.
- Hollandia malt non-alcoholic beer: water, barley, malt, maltose and hop extract.
- Bavaria non-alcoholic beer: pure minerai water, barley malt, maltose and hops.
As control, the flavor profile of a Carlsberg pilsner beer (4.6% (vol/vol) alcohol) was used.
To compare the trial beers brewed with Pichia kluyveri with other commercial pilsner beers, the flavor profile of 2 other pilsner beers was measured: Stella premium lager beer (5.2% (vol/vol) alcohol) and Heineken lager beer (5.0% (vol/vol) alcohol).
As can be seen from the list above, the main ingrédients for the alcohol-free beers are water, malt (sometimes with unmalted barley as adjunct) and hops. These are the same main ingrédients as in the trials with Pichia kluyveri.
The resuit 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 sealed so that the concentration of the respective flavor compound for the Carlsberg pilsner beer is 1.
Figure 2 clearly shows that ail the commercial alcohol-free beers hâve a very low concentration of typical beer flavor compounds, when compared to the Carlsberg pilsner beer and compare to the other three pilsner beers. However, the beer produced with Pichia kluyveri has similar levels of spécifie beer flavor compounds as the reference pilsner beer, w' in particular isoamyl alcohol, ethyl hexanoate and ethyl octanoate. Ethyl butyrate was présent in lower amount (half the concentration) and isoamyl acetate was présent in higher amounts (double or more concentration). When compared to the other pilsner beers, it seems that the isoamyl acetate concentration in the Pichia kluyveri beer is still higher, as well as ethyl octanoate concentrations. However, ethyl butyrate and ethyl hexanoate concentrations were higher in the pilsner beers than in the Pichia kluyveri beer. These compounds are présent in very low concentrations in beer and are less important compared to isoamyl acetate concentrations. Isoamyl alcohol concentrations does not differ much between the Pichia kluyveri beer and the pilsner beers, but was virtually absent in the alcohol-ffee beers. When the overall flavor profile of the Pichia kluyveri beer is compared to to the alcohol-ffee and pilsner beers, it seems that the flavor profile of the Pichia kluyveri beer is closer to a beer with 4.6-5.2% (vol/vol) alcohol than the flavor profile of any of the commercial alcohol-ffee beers measured.
As these flavor compounds hâve a similar ffuity flavor, they work in synergy. Tasting of the Pichia kluyveri beer revealed that the flavor was very beer-like and this beer was preferred over the commercial alcohol-ffee beers by a tasting panel consisting of brewers and beer consumers.
Low-alcohol beer produced with Pichia kluyveri
This beer was brewed from 1500 liter wort and the fermentation température was 21°C (see Figure 1 for brewing process details). This trial was carried out with Pichia kluyveri strain A. Ail the glucose was consumed at the end of fermentation and the final éthanol level was 0.7%. The fermentation time was three weeks.
As can be seen from Figure 3, both the alcohol-ffee and low-alcohol beer prepared with Pichia kluyveri strain A hâve a similar flavor profile, meaning that both the alcohol-ffee and low-alcohol beer are very close to a normal pilsner beer flavor profile. Again the main esters isoamyl acetate, ethyl butyrate, ethyl hexanoate, ethyl octanoate and isamyl alcohol détermine the flavor profile.
Measurement of diacetyl produced by Pichia kluyveri
To be sure that Pichia kluyveri does not produce any signifïcant off-flavors in beer, the production of the most important off-flavor in beer, diacetyl, was measured in a lab brewing trial with Pichia kluyveri strain A only, compared to a beer with a Saccharomyces cerevisiae beer strain (Figure 4).
It is clear from Figure 4 that Pichia kluyveri produces much less diacetyl compared to a Saccharomyces cerevisiae brewing strain. Thjs is important as diacetyl is ofiten regarded as an off-flavor in beer. Especially, in alcohol-free and low-alcohol beers where no alcohol is présent, the taste threshold level of diacetyl is probably much lower than in normal strength beers.
Conclusion
The flavor profile results of the alcohol-free and low-alcohol beers produced with Pichia kluyveri show that this yeast strain is ideally suited for the production of beers with lower or no alcohol. Especially, the production of a high amount of esters, coming from a very low glucose concentration is a spécial property seen in Pichia kluyveri strains. Together with isoamyl acetate, other esters are enhanced, which gives the beers with low or no alcohol a beer-like flavor profile.
Example 2
Saccharomycodes ludwigii has been mentioned in Branyik et al. (2012) as the most successful genus, other than Saccharomyces, used for the industrial production of alcoholfree beer.
To compare the properties of Pichia kluyveri with the properties of Saccharomycodes ludwigii for préparation of low-alcohol beer a second experiment was set up: Materials and methods
Culturing yeast
The two yeast cultures (Pichia kluyveri strain A and a Saccharomycodes ludwigii yeast strain) were prepared in YPD (2% glucose, 1% peptone and 0.5% yeast extract), with an adjusted pH of 5.5. The YPD medium was autoclaved at 121°C for 20 minutes.
The yeast cultures were plated out on YGC plates (Sigma-Aldrich) and a single colony was inoculated in 5 ml YPD medium. These cultures were grown ovemight in a shaking incubator at 30°C and inoculated in 500 ml YPD. After 2 days, a sample was taken for cell counts to be able to inoculate the yeast cultures at the same initial cell count in the wort medium.
Lab brewing trial
Lab-scale fermentation trials were carried out in 500 ml of wort. The wort was prepared with pilsner malt extract (Brewferm). The malt extract was mixed with water to reach an initial sugar content of 7°P (Plato) as measured with an Anton Paar Beer Alcolyzer. In total, 10 liter wort was prepared in this way.
In the first set-up, the wort was boiled for 60 minutes without addition of hops. In the second set-up,
4.5 g of Amarillo, 4.5 g of Cascade and 2 g of Tettnanger hop pellets were added to the wort and boiled for 60 minutes to give bittemess. 10 minutes before the end of boiling, 1.5 g Cascade, 1.5 g Amarillo and 1 g Tettnanger hop pellets were added as aroma hops.
After boiling, the wort was transferred to 2L bottles (1600 ml of wort per bottle), which were closed by water locks.
The yeast cultures were inoculated at lxlO6 CFU/ml and fermentation was carried out at 20°C. After 5 days of fermentation, a sample was taken for flavor analysis.
Ethanol analysis
Ethanol was measured with the Ethanol Enzymatic Bioanalysis kit of Boehringer Mannheim.
Flavor analysis
Flavor analysis was carried out at Laboratorio de Anâlisis del Aroma y Enologia (Zaragoza, Spain). Minor aroma compounds are expressed in pg/L (ppb) and are measured with solid-phase extraction and Gas chromatography with mass spectrométrie détection, while major aroma compounds are expressed in mg/L (ppm) and are measured with gas chromatographic-flame ionization détection after extracting the compounds in dichloromethane. vA
Results
Lab-scale fermentations were carried out in two types of pilsner wort: wort without the addition of hops and the same pilsner wort with addition of bitter and aroma hops (Cascade, Amarillo and Tettnanger) (see materials and methods). Two different yeast strains were tested: Saccharomycod.es ludwigii and Pichia kluyveri. The 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 (P. kluyveri w/o hops)
4) Pichia kluyveri in wort with hops (P. kluyveri with hops)
Ail fermentations were carried out for 5 days at 20°C.
During the fermentation, cell counts were measured every day to follow yeast growth (Figure 5).
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 during the whole fermentation.
After 5 days, éthanol concentration was measured in ail fermentation products (Table 3).
Table 3 : Ethanol concentration in ail 4 fermentation products
| Fermentation product | Ethanol % /v/v) |
| 5. ludwigii w/o hops | 0.3 |
| 5. ludwigii with hops | 0.3 |
| P. kluyveri w/o hops | 0.1 |
| P. kluyveri with hops | 0.1 |
Table 3 shows that both Saccharomycodes ludwigii and Pichia kluyveri produce very low amounts of éthanol. Pichia kluyveri seems better at producing no alcohol, as this strain yy— grows to higher cell counts, but produces less alcohol, compared to Saccharomycodes ludwigii.
The fermentation products at day 5 were also analyzed for flavor profile (Figures 6, 7, 8 and 9). The most important flavor compounds for beer are esters and higher alcohols.
Figures 6 and 7 show the ester concentrations in ail final fermentation products, as well as in the basic wort with and without hops. Ail ester concentrations are the highest in the fermentation products with Pichia kluyveri, both with and without hops. In the case of isoamyl, phenylethyl, isobutyl and butyl acetate, Pichia kluyveri produces higher amounts of these esters when no hops are added, compared to ethyl decanoate. Especially the levels of isoamyl acetate and phenylethyl acetate are very high. Isoamyl acetate results in a banana aroma, while phenylethyl acetate results in a more flowery, honey aroma. Both ester compounds are wanted compounds in beer. It has to be noted that the results depicted here are ester concentrations after 5 days of fermentation, and these concentrations will decrease over time in a final beer. It is important to hâve high ester concentrations to start with, resulting in a fruity beer for a longer period of time, as these ester compounds are hydrolyzed over time in the final beer.
The fusel alcohol concentrations, depicted in Figure 8, are quite similar between the fermentation products of Saccharomycodes ludwigii and Pichia kluyveri. Only isoamyl alcohol concentrations are higher in the fermentation products with Saccharomycodes ludwigii, probably because this compound is not converted to isoamyl acetate, as is the case in the Pichia kluyveri fermentation products.
In Figure 9, three acids are depicted, which are négative flavor compounds in beer. Especially decanoic acid gives a rancid, cheesy flavor to beer and is therefore unwanted. It is very clear that especially Saccharomycodes ludwigii is producing high amounts of these acids, making it less suitable for beer production.
Conclusion
The lab brewing trial with the two different won-Saccharomyces yeast strains clearly indicates that Pichia kluyveri is better suited for the production of alcohol-free beer, compared to Saccharomycodes ludwigii. Pichia kluyveri produces less alcohol, more wanted ester compounds and less unwanted acids, compared to Saccharomycodes ludwigii.
DEPOSITS
The Pichia kluyveri PK-KR1 (JT1.28 or strain A) and PK-KR2 (JT3.71) strains were deposited on 24 August 2006 at the National Measurement Institute, 51-65 Clarke Street, South Melbourne, Victoria 3205, Australia, by University of Auckland, School of Biological Sciences, Auckland 1142, New Zealand, and given the 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 e Silva, J.B., 2012. A review of methods of low alcohol and alcohol-ffee 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: 186-190.
Montanari, L., Marconi, O., Mayer, H., Fantozzi, P., 2009. Production of alcohol-free beer. In: Preedy, V.R. (Ed.), Beer in Health and Disease Prévention. 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 3:165-77.
Stewart, G. Wort glucose, maltose or maltotriose - do brewer’s yeast strains care which one? Institute of Brewing and Distilling. Asia Pacifie Section. 31st Asia Pacifie
Convention 2010. (http://www.ibdasiapac.com.au/asia-pacific-activities/convention- proceedings/2010/results .html) ./j''
Swiegers, J.H., S.M.G. Saerens, I.S. Pretorius. 2007. The development of yeast strains as tools to adjust the flavor of fermented beverage to market spécifications. Havkin D. and Belanger F. Biotechnology in flavor production. Blackwell Publishing Ltd.
Zufall C, and Wackerbauer K. 2000. Process engineering parameters for the dealcoholisation of beer by means of falling film évaporation and its influence on beer quality. Monatsschrift fur Brauwissenschaft 53:124-137. y/''
Claims (8)
- AMENDED CLAIMS1. A method of preparing a low-alcohol or alcohol-free beverage with an alcohol content of no more than 1.2% (vol/vol) comprising the steps ofa) providing a wort; andb) fermenting the wort with at least one Pichia kluyveri yeast strain to obtain the low-alcohol or alcohol-free beverage.
- 2. The method according to claim 1, further comprising step al) adding to the wort at least one hop variety prior to fermenting the wort with the at least one Pichia kluyveri yeast strain.
- 3. The method according to any of the preceding claims, wherein the fermentation of the wort with the at least one Pichia kluyveri yeast strain is carried out for at least 7 days.
- 4. The method according to any of the preceding claims, wherein the fermentation of the wort with the at least one Pichia kluyveri yeast strain is carried out at a température of between 18-22°C.
- 5. The method according to any of the preceding claims, further comprising the stepc) bottling and bottle pasteurizing the low-alcohol or alcohol-free beverage.
- 6. The method according to any of the preceding claims, wherein the at least one Pichia kluyveri yeast strain is capable of fermenting glucose but not maltose or maltotriose.
- 7. The method according to any of the preceding claims, wherein the at least one Pichia kluyveri strain is selected from the group consisting of the Pichia kluyveri PK-KR1 (JT1.28 or strain A) strain that was deposited on 24 August 2006 at the National Measurement Institute, 541-65 Clarke Street, South Melbourne, Victoria 3205, Australia, by University of Auckland, School of Biological Sciences, Auckland 1142, New Zealand, under accession numbers V06/022711, and the yyPichia kluyveri PK.-KR2 (JT3.71) that was deposited on 24 August 2006 at the National Measurement Institute, 541-65 Clarke Street, South Melbourne, Victoria 3205, Australia, by University of Auckland, School of Biological Sciences, Auckland 1142, New Zealand, under accession numbers V06/022712.
- 8. The method according to any of the preceding claims, wherein the low-alcohol or alcohol-free beverage is a beer.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP13158261.1 | 2013-03-07 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| OA17477A true OA17477A (en) | 2016-12-30 |
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