CZ309608B6 - Yeast strain Cyberlindnera suaveolens CCM 9179 and its use in producing non-alcoholic beer - Google Patents

Abstract

The invention relates to the yeast strain Cyberlindnera suaveolens CCM 9179 isolated from the collection strain RIBM Spk 8, which aerobically processes glucose, sucrose, cellibiose, raffinose, xylose, glycerol and lactic acid, does not process maltose, and produces ovoid, ellipsoid or spherical 4 x 4 to 6 μm cells that can form pseudomycelium. The invention also relates to producing non-alcoholic beer, in which the main fermentation of the wort takes place with the yeast strain Cyberlindnera suaveolens CCM 9179.

Description

Yeast strain Cyberlindnera suaveolens CCM 9179 and its use in the production of non-alcoholic beer

Field of technology

The invention relates to a new yeast strain Cyberlindnera suaveolens CCM 9179.

The invention also relates to the use of this strain of yeast in the production of non-alcoholic beer.

Current state of the art

The production of beer consists in the controlled fermentation of the sugars contained in the wort by brewer's yeast. Cultivated yeasts of several species and genera are referred to as brewer's yeast: Saccharomyces pastorianus is used for the production of bottom-fermented beers, Saccharomyces cerevisiae for the production of top-fermented beers, and for the production of special types of beer, e.g. the genera Brettanomyces or Torulaspora (see e.g. T . Kochláňová et al.: "Non-Saccharomyces yeasts and their importance in the brewing industry. Part I - Brettanomyces (Dekkera)" and "Non-Saccharomyces yeasts and their importance in the brewing industry. Part II", both Kvasný Průmys 62/2016 ( 7 to 8)). During their activity, brewer's yeast converts carbohydrates into ethanol, carbon dioxide and the energy needed for their reproduction, as well as approximately two thousand other substances, especially higher alcohols, esters, aldehydes, etc., which affect the resulting sensory properties of the finished beer. The overall ratio of all these higher alcohols and esters is very important for the overall sensory impression, as the individual compounds themselves are very often below the threshold concentration of sensory perception. It is generally known, for example, that strains of the upper yeast Saccharomyces cerevisiae produce a larger amount and a wider spectrum of sensory active substances than strains of the lower yeast Saccharomyces pastorianus (see e.g. Lodolo et al.: "The yeast Saccharomyces cerevisiae - the main character in beer brewing. " FEMS Yeast Res 8(7):1018-36, 2008), and also that the "lighter" taste of bottom-fermented beers causes, in addition to the lower temperature of the main fermentation, also the changed expression of the genes responsible for the production of esters in the species Saccharomyces pastorianus (see e.g. Procopio et al.: "Function and regulation of yeast genes involved in higher alcohol and ester metabolism during beverage fermentation", European Food Research and Technology 233(5):721-729, November 2011).

Non-alcoholic beer is defined as a fermented beverage made from grain malt with an alcohol content of up to 0.5% by volume. Currently, it is produced in several ways: 1) by removing alcohol from already produced beer using temperature or membrane processes, 2) prematurely terminated fermentation, 3 ) by using a special strain of yeast, 4) by changing the mashing conditions (i.e. by adjusting the spectrum of fermentable sugars in the wort), 5) by the continuous fermentation technique; possibly by combining some of these procedures (e.g. using a special yeast strain with reduced alcohol production in combination with premature termination of fermentation, etc.) - see e.g. Brányik et al.: "A review of methods of low alcohol and alcohol-free beer production ”, J. Food Engin. 108: 493-506, 2012. As special yeast strains with reduced alcohol production, e.g. Saccharomyces cerevisiae, Saccharomycodes ludwigii or Zygosaccharomyces rouxii are used, which have a limited ability to ferment the sugars present and produce less alcohol (see e.g. Sohrabvandi et al.: " Characteristics of different brewer's yeast strains used for non-alcoholic beverage fermentation in media containing different fermentable sugars". Iran. J. Biotechnol. 8: 178-185, 2011). In addition, the use of the yeast of the genus Pichia, Candida shehatae (see e.g. Michel, M„ Meier-Dornberg, T„ Jacob, F„ Methner, F.-J„ Wagner, R. S„ and Hutzler, M. (2016 ): "Review: Pure non-Saccharomyces starter cultures for beer fermentation with a focus on secondary metabolites and practical applications". J. Inst. Brew., 122: 569 to 587. doi: 10.1002/jib.381.), Torulaspora delbrueckii (see e.g. Canonico et al.: "Torulaspora delbrueckii in the brewing process: A new approach to enhance bioflavour and to reduce ethanol content", Food Microbiol. 56: 45-51) or Kluyveromyces (see e.g. Medeiros et al.: "Aroma compounds

- 1 CZ 309608 B6 produced by Kluyveromyces marxianus in solid state fermentation on a packed bed column bioreactor", World. J. Microbiol. Biotechnol., 17: 767 to 771). In addition, the use of Williopsis saturnus (a synonym of Cyberlindnera suaveolens) in the production of beer with a reduced alcohol content is known (see, for example, the study Liu, S. Q., & Quek, A. Y. (2016): "Evaluation of Beer Fermentation with a Novel Yeast Williopsis Saturnus" .Food technology and biotechnology, 54(4), 403 to 412). Beers produced in this way can taste like wort, tend to be sweeter and fruitier than alcoholic beers produced in the traditional way or de-alcoholized by physical methods.

The essence of the invention

The essence of the invention is a new yeast strain Cyberlindnera suaveolens CCM 9179, which was isolated from the collection strain RIBM Spk 8. It was prepared for further use by selection and repeated inoculation on wort agar with tetracycline. Cultivation took place aerobically in a microbiological incubator at a temperature of 26 °C for 3 to 5 days. After the growth of the yeast culture on the surface of the agar, this culture was transferred with a sterilized inoculation loop to a new Petri dish with agar and inoculated by the so-called cross spreading, during which the concentration of yeast on the agar is diluted. This procedure was repeated until the yeast culture grew into visibly separate colonies. The subsequent isolation of the yeast strain Cyberlindnera suaveolens CCM 9179 was then carried out by inoculating the selected colony on a Petri dish containing wort agar.

The strain thus isolated was subsequently characterized by biochemical tests and sequencing of the ITS DNA region.

The yeast strain Cyberlindnera suaveolens CCM 9179 produces ovoid, ellipsoid or spherical cells measuring 4 μm x 4 to 6 pm. They multiply vegetatively by multilateral budding, they can produce pseudohyphae (pseudomycelium).

Appearance and consistency of colonies - cream-colored, with wavy edges. The nature of the growth in a liquid medium - in liquids it forms a sediment, slime can form. The strain grows at temperatures below 15 °C, it does not grow at a temperature of 37 °C.

The strain Cyberlindnera suaveolens CCM 9179 aerobically utilizes (oxidizes) glucose, sucrose, cellibiose, raffinose, xylose, glycerol, lactic acid; anaerobically none of them. Does not use maltose.

The yeast strain Cyberlindnera suaveolens CCM 9179 has some advantageous properties for use in the preparation of non-alcoholic beer compared to the existing brewing yeasts Saccharomyces cerevisiae and Saccharomyces pastorianus. In particular, it is the inability to ferment maltose, the main sugar in brewer's wort, due to which this strain produces only low concentrations of alcohol. The optimal temperature for the production of non-alcoholic beer using this strain is between 7 and 9 °C, when the yeast produces the optimal amount of sensory active substances. The optimal degree of wort for the production of non-alcoholic beer is then in the range of 5 to 9% by weight, when the yeast produces the optimal amount of alcohol (up to 0.5% by volume, which is the upper limit of non-alcoholic beer).

The yeast strain Cyberlindnera suaveolens CCM 9179 according to the invention is stored in the collection of microorganisms of the Research Institute of Brewing and Malting, a.s., coded RIBM (Research Institute of Brewing and Malting) under the number RIBM 196 and in the Czech Collection of Microorganisms in Brno under by CCM number 9179.

Examples of implementation of the invention

Example 1: Preparation of the yeast strain Cyberlindnera suaveolens CCM 9179 for laboratory fermentation tests

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In the preparation of the yeast strain Cyberlindnera suaveolens CCM 9179 for practical use - e.g. for its further testing or for the production of non-alcoholic beer, a pure culture of this strain was used, which was obtained by isolation from the spontaneous fermentation of grape must (see above), and which until kept in the refrigerator at a temperature of 2 ±1 °C during preparation.

A culture of the yeast strain Cyberlindnera suaveolens CCM 9179 was inoculated onto a wort agar slant in cotton-plugged tubes. A part of the biomass was then transferred from the created yeast growth to 10 ml of 10% sterile wort using a sterile inoculation loop, after which cultivation was carried out for 2 days at a temperature of 25 °C. Then the upper fermented layer was poured off and the yeast sediment was transferred to 50 ml of 10% sterile wort, after which cultivation was carried out for 2 days at a temperature of 20°C. Subsequently, this strain was multiplied by converting to an ever larger volume in a ratio of approximately 1:4. The yeast sediment was thus first transferred to 250 ml of 10% sterile wort, where propagation took place for 5 days at a temperature of 15 ±1 °C, then the formed yeast sediment was transferred to 1000 ml of 10% wort, where cultivation took place for 3 days at a temperature of 12 ±1 °C.

The yeast strain Cyberlindnera suaveolens CCM 9179 prepared in this way can be directly used for laboratory fermentation tests or for the production of non-alcoholic beer. If necessary, it is previously washed with water (repeated sedimentation and washing), which removes the culture medium from it.

Example 2: Preparation of non-alcoholic beer under laboratory conditions using the yeast strain Cyberlindnera suaveolens CCM 9179

A centrifuged yeast concentrate of Cyberlindnera suaveolens CCM 9179 prepared according to the procedure described in Example 1 was added to the wort (3 liters) with an original extract of approximately 6%. The yeast dose was approximately 7x10 ⁷ cells/ml of wort. Subsequent fermentation took place in a sterilized plastic container at a temperature of 8 °C ±1 °C for 3 days.

After fermentation, the beer prepared in this way was transferred to lager containers and stored in the lager cellar at a temperature of 2 ± 1 °C. After 3 weeks of lying down, his alcohol content and bitterness were measured. The measured values are shown in Table 1.

Table 1. Basic chemical analysis

Measured parameter unit value Alcohol % vol. 0.13 Original wort extract (Plato) wt% 7.87 Bitterness (isocompounds) units of bitterness 9.5

As can be seen, the strain Cyberlindnera suaveolens CCM 9179 produces only low concentrations of alcohol compared to common brewer's yeast.

Furthermore, a sensory evaluation of the non-alcoholic beer prepared in this way was also carried out by a tasting committee. This beer showed no sensory defects. The overall subjective impression was evaluated as very good. The beer differs from available non-alcoholic beers with notes of citrus and honey in the aroma and taste.

Example 3: Preparation of non-alcoholic beer using the yeast strain Cyberlindnera suaveolens CCM 9179

A centrifuged yeast concentrate of Cyberlindnera suaveolens CCM 9179 prepared according to the procedure described in Example 1 was added to the wort with an original extract of approximately 8.5%. The yeast dose was approximately 7x10 ⁷ cells/ml of wort. Subsequent fermentation took place in a sterilized plastic container at a temperature of 7°C ±1°C for 5 days.

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After fermentation, the beer prepared in this way was transferred to lager containers and stored in the lager cellar at a temperature of 2 ± 1 °C. The beer was kept in lager containers for 4 weeks. It was then measured for fermentation, extract, alcohol content and bitterness. The measured values are shown in Table 2.

Table 2. Basic chemical analysis

Measured parameter unit value Extract apparent wt % 8.29 Extract real wt% 8.34 Alcohol wt % 0.09 Alcohol % vol. 0.12 Original wort extract (Plato) wt % 8.51 Apparent fermentation % 2.58 The real fermentation % 2.06 Bitterness (isocompounds) units of bitterness 9.4

Furthermore, a sensory evaluation of the non-alcoholic beer prepared in this way was also carried out by a tasting committee. The results are presented in Table 3. The dominant taste was evaluated as sweet and 10 fruity (citrus); the beer also had a very weak yeasty, caramel and burnt taste.

A non-alcoholic beer produced using the strain Cyberlindnera suaveolens CCM 9179 was evaluated as different from the available non-alcoholic beers - citrus with notes of honey.

Table 3. Sensory evaluation

Monitored parameter Evaluation zest 1.9 fullness 1.3 bitter 0.4 bitterness - culmination 1.1 bitterness - fading 0.3 bitterness - character 1.3 bitterness 0.4 sweet taste 3.3 sour taste 0.7 hop flavor 0.6 fruity/ester taste 1.7 malty taste 1.6 yeasty taste 0.4 wort taste 1.7 malt flavor 2.5 perfume taste 0.8 diacetyl taste * caramel flavor 1.0 burnt taste * Suits the style 1.5 Balance 2.4 Popularity 4.2 Overall impresion 3.0

* not recorded

Claims (2)

1. The yeast strain Cyberlindnera suaveolens deposited at the Czech Collection of Microorganisms under the number CCM 9179, isolated from the collection strain RIBM Spk 8, which aerobically processes glucose, 5 sucrose, cellibiose, raffinose, xylose, glycerol and lactic acid, does not process maltose, and produces ovoid, ellipsoid or spherical cells measuring 4 x 4 to 6 μm. 2. A method of producing non-alcoholic beer, characterized in that the main fermentation of the wort takes place with the yeast strain Cyberlindnera suaveolens CCM 9179 according to claim 1.