The present invention concerns a method of managing yeast and dispatching the yeast to process sites, in particular for the production of fermented beverages.

BACKGROUND TO THE INVENTION

At present, yeast cultures are propagated and transported in the form of so-called yeast slopes. A drawback of the present managing method is that the yeast slopes may contain a rather large number (up to 5% or even more) of yeast cells having defects in their respiratory chain. Pitching to a wort or fermentable broth with a yeast culture comprising such defect yeast cells (petites), may result in a slowed down fermentation or typically may result in a higher production of off-flavours during fermentation, increasing beer maturation time at the end of fermentation and/or result in an undesired flavour profile of the fermented beverage and/or flocculate at an undesired time or rate during the fermentation process.

As the number of petites tend to increase during reuse of yeast batches in subsequent fermentation cycles, yeast slopes with a high number of petites, cannot guarantee high quality brews during a number of consecutive fermentation cycles, which results in an important cost increase for brewing.

It is known that petites are defects in the yeast's mitochondrial or chromosomal DNA that occur when the yeast is grown under elevated stress conditions. The yeast management system in, for example breweries, comprises several process steps wherein yeast cells are under high stress conditions. In particular, in companies producing fermented beverages in a worldwide network of breweries, yeast cultures are typically kept in a central yeast collection that, on a regular basis, dispatches the yeast cultures to the different breweries.

At the central yeast collection, the yeast cultures are preserved by freezing and storage at a temperature of minus 80°C or lower. For dispatch, the yeast cultures are subsequently thawed, propagated and transferred to a nutrient medium (agar) in a vial or container (the so-called yeast slopes).

During the freezing and thawing of the yeast culture and even subsequent propagation and transportation steps, free oxygen radicals can be formed which are known to cause DNA mutations and may impact the respiration chain of the yeast cells, potentially resulting in yeast slopes having a high amount of yeast cells having deficient functional respiratory chain and negatively impacting the brewing process in the breweries. The present invention addresses the above problems and allows guaranteeing the quality of yeast cells in terms of viability and fermentation capability at the time of dispatch of the yeast slopes.

SUMMARY OF THE INVENTION

The present invention concerns a method of yeast management comprising preserving and propagating yeast cells at a central yeast collection facility and dispatching yeast cells from that yeast collection facility to distant yeast processing sites, the yeast management method comprising:

(a) Preserving yeast cells at a temperature of minus 80°C or less;

(b) Thawing said yeast cells under conditions allowing revitalisation of a majority the yeast cells;

(c) Propagating the thawed yeast cells;

(d) Packing at least part of the propagated yeast cells for transport to yeast processing sites;

(e) Production of a fermented beverage by inoculating a fermentable broth with yeast cells propagated from the packed and transported yeast cells of step (d);

(f) Optionally preserving part of the propagated yeast cells by freezing the yeast cells to a temperature of minus 80°C or lower,

Characterized in that the method comprises a step of: (g) growing the yeast cells on an exclusively non-fermentable carbon source to selectively propagate yeast cells having a regularly functional respiratory chain (non- petites). Preferably, the invention comprises an additional step of conditioning the yeast cells for preservation.

Method step (f) can be performed at any time during the above method such as after thawing of the yeast cells; during propagation of the thawed yeast cells; before, during or after propagation of the yeast cells; prior to or during the conditioning; or prior to the optional freezing of the yeast cells.

In method step (f) the yeast cells are grown on the exclusively non-fermentable carbon source for a period allowing at least 3 doubling cycles of the yeast cells having a regularly functional respiratory chain. The non-fermentable carbon source is hereby preferably selected from the group comprising: glycerol, lactate, ethanol and/or admixtures thereof.

Prior to freezing of the yeast (culture) for preservation, the yeast is preferably propagated on a carbon source comprising maltose and trehalose and/or may be treated with RNAIater or Allproctect.

During preservation, the yeast cells may be in contact with a medium comprising a non- fermentable carbon source; on a medium comprising zinc; a medium comprising an oxygen scavenger and/or an antioxidant (eg. glutathion); a medium comprising 15 to 80w%, preferably 60 to 80w%, glycerol as cryoprotectant.

The yeast cells are preferably packed in a vial or container containing an agar comprising a carbon source, whereby the yeast cells are not provided as single clone forming colonies (at no point single clones are selected). This is especially the case when the yeast cells form a complex culture. The agar comprising a fermentable carbon source, preferably contains a yeast-malt broth (eg. 3g/L yeast extract, 3g/L malt extract, 5g/L peptone and 10 g/L dextrose) The vials or containers may be hermetically sealed after introduction of the yeast cells. Optionally an oxygen scavenger is provided in said vial or container. The present invention also concerns vials or containers containing a yeast culture for transporting the yeast cells in accorda nce with the method described supra. The yeast culture in said vial or container comprising at least 99% of yeast cells having a regularly functional respiratory chain (max. 1% petites). Preferably, the vial or container according to the invention is made from or coated with a material having a permeability for radiation with a wavelength ranging from lOnm to 400nm of Iess than20%.

The method and vials or containers according to the present invention are believed to be particularly suitable for dispatching yeast cultures for the preparation of a fermented beverage at the yeast processing site.

Fina lly, the yeast culture, preferably comprises yeast of a Saccharomyces sp. BRIEF DESCRIPTION OF THE DRAWINGS

Fig. la A culture (control) containing both normal (red, grey in the figures) as well as respiratory deficient (white, small) cells have different phenotypes when plated on a YPD - plate (Yeast extract 1%; Peptone 2%; Dextrose 2%; Agarose 2%) overlaid with TTC (2,3,5- triphenyltetrazolium chloride);

Fig. lb The culture prepared by a method according to the present invention plated on the same type of plate as before (Yeast extract 1%; Peptone 2%; Dextrose 2%; Agarose 2%) and overlaid with TTC (2,3,5-triphenyltetrazolium chloride). DETAILED DESCRIPTION OF THE INVENTION

The viability of yeast (eg. Saccharomyces cerevisiae) in a beverage fermentation process is a key feature for optimization of the conversion of sugars into ethanol and for optimizing production of desired flavour compounds essential for the organoleptic signature of the resulting beverage.

I n accordance with the present invention, yeast viability and yeast quality can be ensured. In beer production, yeasts are typically stored (preserved) for long term in a freezer at temperatures of -80°C or lower. The yeast preservation can be achieved by first conditioning the yeast cells and subsequently freezing the conditioned yeast cells. An example of yeast preservation comprises the steps of, aseptically inoculating a yeast in a sterile brewers wort and subsequently propagating the yeast in aseptic conditions. After propagation a, preferably small, sample of the yeast is transferred to a cryovial and mixed with a cryoprotectant solution, such as a 15-80w%, preferably 60-80w% glycerol solution. The cryovial is subsequently closed and stored in a freezer at -80°C or lower.

As freezing of the yeast cells and later thawing of the yeast cells are best performed at high rate, it is preferred to use cryovials of limited volume (eg. 4ml).

An example of yeast preservation comprises aseptically inoculating yeast (eg. from an agar medium, optionally comprising treha lose and/or maltose) in an autoclaved 500 ml Schott bottle filled with 90ml of sterile, 13,5°Plato brewers wort. The bottle is closed with a waterlock and incubated on a shaking platform at 105 rpm in an incubator at 19°C. After 42 hours of incubation (yeast propagation), a 2.1 ml sample of the yeast is added to a 4 ml sterile cryovial (eg. Cryoclear™ by Globe Scientific Inc.) to which 0,9 ml of a sterile 73%v/v glycerol-solution is added after which both are mixed. After mixing, the cryovial is stored at a temperature of -80°C for preservation.

As yeast cultures used in beverage fermentation processes will evolve during subsequent fermentation cycles, it is necessary to sta rt from a 'fresh' yeast culture for fermentation after a given number of brewing cycles (typically after 4, 5 or 6 fermentation cycles) to limit the chance of brewing a batch of fermented beverage that has a non-desired organoleptic profile.

The 'fresh' yeast can either be propagated starting from a thawed yeast removed from the freezer or starting from a yeast culture propagated from a sample of propagated thawed yeast removed from the freezer.

As in larger breweries, yeast preservation is centralized, fresh yeast samples need to be dispatched to several breweries at regular time intervals and yeast viability in these samples needs to be ensured throughout the entire yeast management system from preservation until fermentation.

In order to ensure or even improve yeast viability during the subsequent parts of the yeast management system, the present invention provides a method including:

i. Thawing said yeast cells under conditions allowing revitalisation of a majority the yeast cells;

ii. Propagating the thawed yeast cells;

iii. Packing at least part of the propagated yeast cells for transport to yeast processing sites;

iv. Production of a fermented beverage by inoculating a fermentable broth with yeast cells propagated from the packed and transported yeast cells of step (iii);

v. Optionally preserving part of the propagated yeast cells by freezing the yeast cells to a temperature of minus 80°C or lower,

whereby in accordance with the present invention, the method comprises a step of:

vi. growing the yeast cells on an exclusively non-fermentable carbon source to selectively propagate yeast cells having a regularly functional respiratory chain (non-petites).

Growing the yeast cells on an exclusively non-fermentable carbon source such as glycerol, lactate or ethanol, allows propagating only those yeast cells that have a regularly functional respiratory chain and hence can use non-fermentable sugars as carbon source, which is not the case for yeast cells having defects in their mitochondrial DNA. As an example of the above method of the invention, a cryovial stored at -80°C is removed (starting from the storage and preservation example described supra) from the freezer and thawed by placing it in a waterbath at 31°C. As the cryovial preferably only contains a limited volume of yeast solution (eg. 3 ml), thawing at high rates is achieved.

Next a sample (eg. 900 microliter) of the cryovial content is transferred to an autoclaved Schott bottle filled with 90ml of sterile, 13,5°Plato brewers wort. The bottle is closed with a waterlock and incubated on a shaking platform at 105 rpm in an incubator at 19°C. After 42 hours of incubation, 900 microliter of the yeast culture is transferred to an autoclaved 500 ml Schott bottle filled with 90 ml ml of sterile YPGIycerol medium (yeast extract 1% w/w; Peptone 2% w/w; glycerol 2,5% v/v and water). The bottle is closed by a waterlock and incubated on a shaking platform at 105 rpm in an incubator at 19°C for a time allowing at least 3 doubling cycles of the yeast cells (eg. 66 hours). As the YPGIycerol medium does not contain fermentable sugars, only yeast cells able to retrieve carbon from glycerol will propagate. After 66 hours of incubation the mixture is homogenized thoroughly and incubated on a YM-agar (yeast extract 1% w/w; malt extract 3% w/w; agar 2% w/w and water) in a tube. The tube is then closed with a cotton plug and labelled. The tube (also addressed to as yeast slope) is incubated horizontally for 48 to 72 hours at 24°C, subsequently cooled to 4°C and then shipped in a safe container in a cooled transport to a yeast processing site.

Part of the yeast incubated on the YM-agar is typically used as a starting point for preservation at -80°C or lower as disclosed supra.

In support of the invention, figure 1 shows a yeast culture prepared by method steps i) and ii) as identified above, whilst figure 2 shows a yeast culture starting from the same preservation sample as the culture in Figure 1, but prepared by a method according to the present invention as exemplified here above. It is clear that no respiratory deficient colonies remain after the growth step on YPGIycerol, a medium that as its sole carbon source has glycerol, an exclusively non-fermentable carbon source The yeast processing site can either be a brewery or a laboratory, where the yeast is propagated and subsequently inoculated in a fermentable broth for beverage production or shipped to a brewery for doing the same.

Apart from yeast extract, malt extract, agar and water, the YM-agar may also comprise trehalose and/or cereal rootlets. During transport, the yeast slopes may be closed by a cotton plug or hermetically sealed. Additionally an oxygen scavenger and/or antioxidant such as glutathion may be provided in the tube or agar.

Beer yeast cultures may be well defined yeast cultures or undefined yeast cultures, yet as during the entire yeast management system and during brewing the yeast cells are subjected to several stress factors, it is preferred that during the entire yeast management systems working with single clones (point single clones) is avoided.