GB2584286A

GB2584286A - A process for fed-batch yeast propagation

Info

Publication number: GB2584286A
Application number: GB1907455.8A
Authority: GB
Inventors: Donnelly Daniel
Original assignee: Dandonnellytek Ltd
Current assignee: Dandonnellytek Ltd
Priority date: 2019-05-27
Filing date: 2019-05-27
Publication date: 2020-12-02
Also published as: GB201907455D0

Abstract

Disclosed is a process for the propagation of an alcohol beverage yeast comprising; initiating a batch phase propagation of yeast, maintain until growth rate reduces to a particular level, maintaining aeration of the batch for between 10 and 20 hours, and subsequently adding a feed medium and conducting a fed-batch phase propagation. The fed-batch propagation is conducted at a specific growth rate in the range of 0.08 to 0.18 hr-1. Preferably, the process produces a high cell density yeast which comprises from 1000 to 2500 million cells per ml. Additionally, the acetaldehyde levels at the end of the process can range from not detectable to 37 ppm. Also claimed is the yeast produced from this process, a method of using said yeast to ferment an alcoholic beverage and a growth medium suitable for use in the fed batch stage of yeast propagation.

Description

A process for fed-batch yeast propagation

Field

The present application relates to a process for the propagation of high cell density yeast cultures. The process is particularly useful in the brewing and distilling industries.

Background Of The Invention

The brewing and distilling industries have seen significant growth in recent years. In particular, there has been significant growth in the area of craft brewing and distilling.

In the brewing industry, brewers traditionally re-use yeast cropped from fermentation to pitch subsequent brews. However, this process cannot continue indefinitely due to issues including spontaneous occurrence of respiratory-deficient mutants and an increased risk of contamination. Fresh yeast is therefore propagated at regular intervals. Freshly propagated yeast is introduced into the brewery after approximately 10 fermentation cycles. Large breweries generally propagate their own proprietary yeast strains whereas smaller breweries have to rely on yeast purchased from yeast production companies. This can be a costly process due to the large amount of yeast needed to supply to a fermentation process.

For distilleries, yeast is produced in specialized yeast production units, separate from the distillery, using a fed-batch system usually using molasses as the carbon source for growth. Distilleries do not reuse this yeast and the spent yeast is disposed of in the pot ale fraction as a waste product.

For many years' brewers have used the fact that they use their own yeast to produce their own beers in the establishment of provenance of their beer brands. With craft distilling now mirroring the growth trajectory of craft brewing, there is a growing demand for bespoke yeasts.

There is a commonly held belief that the flavour compounds produced by yeast contribute little to the final overall flavour of the spirit produced. Recently this view has been challenged. Off-flavours produced during the alcohol beverage production process have been found to contribute to the final flavour of the alcohol beverage produced. The quality of the yeast used to pitch the wort during the brewing process is an important factor in achieving a high-quality beer. There is therefore a need for a quality yeast which when used in alcohol beverage production results in an alcohol beverage that is substantially free of certain undesirable off-flavour compounds.

Furthermore, there is a need for an improved process for the propagation of high cell density yeast cultures, in particular, a process that can be used for propagating high-density yeast cultures in-house.

Summary

Accordingly, in a first aspect, the application provides a process for the propagation of an alcohol beverage yeast comprising: (i) inoculating a medium with an alcohol beverage yeast strain and initiating a batch phase propagation to generate yeast at a first yeast growth rate within a batch; (ii) maintaining the batch phase propagation until the first yeast growth rate reduces to a second yeast growth rate; (1) maintaining an aeration of said batch during a period of between 10 and 20 hours from said initiation of said second yeast growth rate; and (iv) subsequently adding a feed medium and conducting a fed-batch phase propagation to generate the alcohol beverage yeast, the fed-batch phase propagation having a third yeast growth rate, the third yeast growth rate being greater than the second yeast growth rate; wherein said fed-batch propagation is conducted at a specific growth rate in the range 0.08 to 0.18 hr' andacetaldehyde levels at the end of the process are less than 37 ppm.

The levels of acetaldehyde at the end of the propagation process can range from undetectable to <37ppm.

Preferably, the acetaldehyde levels at the end of the process are <3 ppm.

The alcohol beverage yeast produced according to the process described herein comprises a high cell density yeast culture comprising from 1,000 to 2,500 million cells per ml.

In accordance with the present teaching, the medium in step (i) comprises malt extract in an amount 122 to 140 g/L, potassium salt in an amount 2 to 3 g/L and zinc ions in an amount 0.1 to 0.2 ppm 7112.. Preferably, the medium comprises malt extract in an amount 132 g/L, potassium dihydrogenphosphate (KH2PO4) in an amount 2.5 g/L and zinc ions in an amount 0.00015 g/L.

The feed medium preferably comprises one or more of the following: malt extract in an amount of 300 to 350 g/L, yeast extract in an amount of 5 to 15 g/L, preferably 10 g/L, and glucose in an amount of 300 to 350 g/L.

The process according to the present application further comprises the step of maintaining the pH in the range 4.9 to 5.1, preferably pH 5.

In accordance with the process of the present application, batch phase propagation is conducted for a period of time in the range 14 to 20 hours.

The fed batch phase is preferably conducted for a period of time in the range 8 to 24 hours at a specific growth rate in the range 0.1 to 0.17 h-1.

In a further aspect, the application provides a method of fermentation for the production of an alcohol beverage comprising use of yeast propagated according to the process described herein. The level of acetaldehyde in the alcoholic beverage produced by the method described is <5ppm, preferably <3 ppm, more preferably <1.25ppm. The alcohol beverage is preferably selected from the group consisting of beer, lager, ale and whisk(e)y.

In a still further aspect, the present application provides a growth medium for use in propagation of an alcohol beverage yeast; said medium comprising: Malt extract in an amount in the range 122 to 140 g/L; Potassium salt in an amount in the range 2 to 3 g/L; Zinc ions in an amount in the range 0.1 to 0.2 ppm Zn2+g/L; and additional nutrients and/or vitamins.

Accordingly, the present application provides a process for the propagation of an alcohol beverage yeast, yeast propagated according to the process described herein, a method of fermentation for the production of an alcohol beverage and a growth medium for use in the propagation of an alcohol beverage yeast, as detailed in the appended claims. Advantageous embodiments are provided in the dependent claims.

The process according to the present teaching will be better understood with reference to the detailed description and examples which follow.

Brief Description Of The Drawings

The present application will now be described with reference to the accompanying drawings in which: Figure 1 shows cell growth optimized in laboratory batch, Medium 1, culture for yeast cell density; Figure 2 shows laboratory batch, Medium 1, followed by fed batch, in medium 4, growth of yeast; Figure 3 shows the development of acetaldehyde and combined higher alcohols levels during and after a batch fermentation; Figure 4 shows the volatile analysis at the end of fermentation of yeast (high density fed-batch propagation) propagated by the process according to the present teaching (experimental yeast) versus that of a fermentation pitched with a cropped yeast; Figure 5 shows levels of volatile compounds at the end of fermentation of a stout for experimental (high density fed-batch propagated yeast) versus control beer (pitched with cropped yeast); and Figure 6 is a schematic of an apparatus for carrying out the yeast propagation process described herein.

Detailed Description

The present application relates to an improved process for the propagation of high-density yeast cultures. The high cell density yeast cultures produced according to the process are particularly suitable for use in the production of beverage alcohol, for example, beer, lager, ale and spirits.

The process described can be used in-house in breweries and distilleries for the propagation of yeast, in particular an alcohol beverage yeast. The process is a fed-batch methodology that is based on use of malt extract with added salts and vitamins. In this context, it will be understood that a fed-batch fermentation process is a process that allows an operator to add one or more nutrients on a continuous or intermittent basis into a fermentation cycle to control the metabolic activity of cells and generate high cell densities.

The term "propagation" as used herein means a process for growth of yeast that results in an increase of an initial yeast cell population.

The term "an alcohol beverage yeast" is used herein to describe yeasts which are suitable for use in the production of alcohol beverages. The person skilled in the art will know that different yeasts can be selected depending on the desired alcoholic beverage to be produced. Suitable yeasts for use in accordance with the present teaching include ale yeast strain such as Lift for example, lager, stout, Weiss bier and distillers' yeast, such as those used to produce grain neutral spirit, and whisk(e)y strains.

It will be appreciated by the skilled person that several factors influence the growth of yeast and are important for a successful yeast propagation process. Key factors which need to be considered when propagating yeast include oxygen, pH, temperature and wort composition.

Diluted molasses supplemented with nitrogen and other minor nutrients are typically the most appropriate growth medium used for propagating yeast in high cell density systems for the production of Bakers' yeast. Baker's yeast will be understood to be of the species Saccharomyces cerevisiae, and is the same species (but a different strain) as the kind commonly used in alcoholic fermentation, which is called brewer's or distiller's yeast. The present application describes a yeast propagation process based on yeast growth in the malt extract sugars, glucose, maltose and maltotriose, rather than sucrose, to grow yeast in a fed-batch system. Ammonia is used as the nitrogen source in the process according to the present teaching.

In one aspect of the present teaching, the carbon source used for the batch process is wort (wash) which is produced in breweries and distilleries. It will be understood that wort is the name given to the liquid extracted from the mashing process during the brewing of beer or whisky. Wort contains the sugars, the most important being maltose and maltotriose, that will be fermented by the brewing yeast to produce alcohol. The use of wort as a carbon source, per the present invention, represents a cost saving for the industry as the carbon source is produced in house and does not have to be bought in.

The process according to the present teaching produces high cell density alcohol beverage yeast. The process is designed such that the entire yeast broth from the fed-batch phase can be used to inoculate or pitch the main (beer) fermentation.

The process described herein demonstrates that the supplementation of malt extract with potassium salts and zinc ions is essential for the growth of a brewing yeast to high cell densities. This, combined with a feedforward control system and high Kea values, typically between 0.03 and 0.04 s-1, allows the attainment of high cell densities in the resulting yeast.

It will be appreciated by the person skilled in the art that the KLa value is the volumetric mass-transfer coefficient that describes the efficiency with which oxygen can be delivered to a bioreactor for a given set of operating conditions.

In a preferred aspect of the present application, the KLa during batch phase propagation is in the range from 0.02 to 0.05 s-1, preferably, 0.03 and 0.04 s-1.

Advantageously, no complex sensors are required during the propagation process described herein, as the feed rate of substrate is determined using a feed-forward control strategy.

In accordance with the present teaching, the feed rate is selected such that the sugar concentration in the stirred tank reactor is kept below 1% sugar. This ensures that there is no Crabtree effect.

In conventional yeast propagation systems batch growth is conducted in single or multiple vessels at a low specific growth rate in order to keep acetaldehyde levels low. This is achieved by having a low aeration rate (low kLa). In such conventional processes the batch phase takes up to 48 hours for completion. Advantageously the batch phase in the process according to the present invention is carried out over a period in the range 14 to 24 hours.

Typical cell densities at the end of conventional yeast propagation processes are approximately 100 million cells per ml. The process according to the present teaching advantageously enables high cell density yeast cultures having a cell density in the range from 1,000 million to 2,500 million cells per ml to be obtained.

In accordance with the process according to the present teaching, the high cell density yeast culture produced may be transferred to a fermentation vessel after a period of between 22 and 44 hours.

The process will be described in more detail below with reference to the examples.

Experimental Yeast Strain and Culture Conditions An ale yeast strain, Lif1, was used in this example. It was maintained on Malt Extract agar (Oxoid) slopes and grown in Malt Extract shake flasks prior to being used as the inoculum for the batch phase of aerobic propagation. Yeast strains were stored on malt extract slopes at 4°C and sub-cultured weekly. Media constituents were optimized in shake flask aerobic cultures grown on an orbital shaker. Yeast viability was determined using the methylene blue staining method.

Laboratory-Scale Fermentation Stirred Tank Reactor (STR) Trials The cultures were grown at 30°C in a 1.8 I laboratory bioreactor (Bioengineering AG, Wald, Switzerland), with a working volume of 1 I, equipped with a double 6-blade Rushton-type agitator, baffles, temperature and pH probes and control mechanisms, air inlet and outlet ports, a base inlet port, a feed inlet port and a sampling port. The outlet passed through a condenser to minimize liquid loss by evaporation. A solution of 4 M NaOH was used to maintain automatically pH 5; no acid control was necessary. The fermentation system was equipped with a gas analyser which measured the level of carbon dioxide and oxygen in the off-gas from the fermenter.

The batch phase of the culture was carried out with an agitation speed of 800 rpm and an inlet air flow rate of 10 I/min (1.25 vvm). The agitation speed was raised to 1,200 rpm during the fed-batch phase in order to enhance oxygen transfer. A detailed account of liquid volumes entering the reactor (base and medium feed) and leaving it (samples) was made in order to have a continuous inventory of the reactor volume throughout the experiment. Samples of 12 ml were taken at regular intervals and cell numbers were determined using a haemocytometer.

Plant-Scale Fermentation Propagation Trials.

Batch medium was sterilized in situ in a stirred tank reactor, STR, for 15 minutes at 121°C. The cultures were grown at 28°C in a STR equipped with a triple 6-blade Rushton-type agitator, dissolved oxygen, temperature and pH probes and control mechanisms, air inlet and outlet ports, a base inlet port, a feed inlet port and a sampling port. A solution of 25 % aqueous ammonia was used to maintain automatically pH 5; no acid control was necessary.

Software Development and Controller Design The controller design used in the process according to the present teaching is based on the feed forward control part of that described by Dabros et a/. (2010) (Michal Dabros, Moira Monika Schuler and Ian W. Marison. Simple control of specific growth rate in biotechnological fed-batch processes based on enhanced online measurements of biomass. Bioprocess Biosyst Eng (2010) 33:1109-1118).

The feed control is based on the fact that yeast cells grow exponentially. Therefore, where xo is the initial cell concentration and x is the cell concentration at a particular time (t) and p is the specific growth rate. (1)

The substrate addition rate, during the fed-batch phase of the fermentation, should logically also follow the same exponential dynamics which can be found by scaling: (2) where Fo is the initial feed needed to support the initial biomass and xe to start fed-batch at desired growth rate (p). The feed rate will also depend on volume at the end of batch phase (V0), as well as the sugar concentration in the feed supply (Sr) and on the biomass yield (5z, ) i.e. the biomass resulting from this sugar utilization. Then the equation for the determination of the initial flow rate of the feed (Fe) is given by: (3) The feed rate at any time during the fed-batch phase, (FFF) can be determined by combining the substrate and biomass balances i.e. by combining equations (1) and (3) to yield the expression: (4) Equation (4) forms the basis for the feed-forward control of the fed-batch phase ofi growth. The specific growth rate (p) and the biomass yield (1r: ) were determined experimentally and a peristaltic pump was used to implement the controller action FFF (t) and supply of feed to the fermentation. Typically, the final volume of the fermentation was comprised of 66.6 % batch and 33% fed-batch volumes. All programs were generated in LabVIEWTM and were coded in-house.

Volatile Analysis Samples were analysed for selected esters, higher alcohols, vicinal diketones and acetaldehyde.

Measured esters were ethyl acetate, isoamyl acetate and 2-phenyl ethyl acetate. Higher alcohols measured were 1-proponal, isobutanol, 2-methyl-1butanol, 3-mithey-1-butanol and 2-phenyl ethanol. The sum of the concentrations of these higher alcohols was expressed as combined higher alcohols in pads per million. Vicinal diketones measured were free and total diacetyl and free and total pentandione-2,3. Acetaldehyde was used as a marker compound for the propagation process.

Results Growth Medium Optimization The constituents of the batch and fed-batch media were optimized using shake flask cultures grown at 30°C. The optimized media are shown in Table 1.

Batch Feed Medium 1 (g/L) Medium 2 (a) Medium 3 (g/L) Medium 4 (a) Wort (solids content) 132 Malt Extract 132 350 Yeast Extract 5 10 Glucose 350 NIKINaHP01 2.5 2.5 K2HPO1 2.5 2.5 (N1-14250, 1 1 MgSO4 x 7H20 0.5 0.5 MnSO4 0.0004 0.0004 Zinc ion (Zn2t) 0.00015 0.00015 Thiamine hydrochloride 0.0004 0.0004 Biotin 0.000002 0.000002 Panthothenic acid 0.0004 0.0004 Table 1. The composition of media optimized for batch and fed-batch fermentations.

Laboratory-Scale Fermentation Stirred Tank Reactor (STR) Trials A typical batch growth was carried out in medium 1 in a laboratory-scale fermentation STR. With sufficient dissolved oxygen, between 90% and100% of the saturation value, and pH, the control and the optimized growth medium, in batch culture this yeast strain was capable of attaining cell densities of in excess of 300 million cells per m1-1 approximately 20 hours after inoculation, Figure 1.

Using a specific growth rate of 0.1 hr-1, cells densities as high as 2,500 cells m1-1 were attained. Figure 2 shows a fed batch laboratory trial in which the cell density reached 1,300 million yeast cells per m1-1 20 hours after commencing the fed batch phase. During the batch then the total biomass, and hence the oxygen demand, increased. It was found to be essential to not allow the oxygen concentration to fall below 60% of the saturation value.

Plant-Scale Fermentation Propagation Trials The optimized laboratory fed-batch process was modified for plant-scale fermentation. NH4OH was used to control pH and wort was used instead of malt extract. The target time was set at 24 hours for the entire propagation process. The batch phase was shortened to 15 hours and the specific growth rate was increased from the 0.1 h-1 to 0.17 h-1 which resulted in a faster feed rate and hence a shorter fed-batch phase.

Volatile Analysis of Laboratory STR Fermentations.

In laboratory experiments acetaldehyde levels at the end of batch phase growth were typically in the region of 200 ppm. At the end of the fed-batch phase in laboratory trials the acetaldehyde was not detectable. In order to determine whether the disappearance of acetaldehyde was due to stripping by air supply to the fermenter or of its re-assimilation by the fed batch grown cells, a batch growth trial was performed where the STR was aerated long after growth has ceased. Figure 3 shows that even in the absence of the fed-batch phase acetaldehyde, and other volatiles compounds produced during propagation, the higher alcohols were lost from the fermentation medium. This demonstrates that air stripping is the mechanism by which volatiles are removed.

The removal of acetaldehyde by stripping the volatile compounds from a batch fermentation does not provide a strategy for the removal of unwanted volatiles from such propagations because, in the absence of a carbon source, yeast cells were seen to rapidly autolyse with large losses in viability being observed. However, and in accordance with the present invention, the provision of a carbon source, during the fed-batch fermentation described herein overcomes any loss in viability. Yeast populations at the end of the fed batch phase were typically 100% viable.

Plant-Scale Fermentation Propagation Trials India Pale Ale (IPA) Trial Certain aspects of the optimized laboratory fed-batch process were modified for this experiment. NH4OH was used to control pH. Medium 4 (see Table 1) which included malt extract was used as the fed-batch medium. The target time was set at 24 hours for the entire propagation process. The batch phase was shortened to 15 hours and the specific growth rate was increased from the 0.1 h-1 to 0.17 h-1. This resulted in a faster feed rate and hence a shorter fed-batch phase. Typical end-of-batch batch yeast cell counts were lower than those for the optimized process, 900 million yeast cells m1-1 versus up to 2,500 million yeast cell m1-1. With a shorter fed batch time the level of acetaldehyde at the end of the fed batch phase was typically in the region of 37 ppm.

A control India Pale Ale (IPA) brew (12.6°F) was pitched with cropped yeast from a previous fermentation. A second brew, the experimental brew, was pitched with the fed batch propagated yeast in a separate fermentation vessel (FV) having the same dimensions as that of the control brew. Both fermentations reached their attenuation limit after 4 days and samples were taken for volatile analysis at the end of fermentation. Ester levels were similar in the experimental versus the control. Combined higher alcohols were similar in the control and the experimental, 224.6 ppm versus 216.2 ppm respectively. The acetaldehyde level was lower in the experimental beer compared to the control (3.0 ppm versus 5.4 ppm). The level of vicinal diketones in the green beers is shown in more detail in Table 2. It will be appreciated by the person skilled in the art that diacetyl is a vicinal ketone which is responsible for a buttery flavour in finished beer while 2,3-pentanedione is a vicinal diketone (VDK) that gives a honey-like flavour to beer.

Control Experimental +/lanai Di ketone free diacetyl 0.04 0.0 total diacetyl 0.04 0.0 free pentanedione-2,3 0.02 <0.01 total pentanedione-2,3 0.02 0.00 Table 2. The level of vicinal diketones at the end of fermentation in green IPA beers.

Although of lesser importance for the 1°1 brew, as the vicinal diketone level is generally very well controlled within a brewery, similar levels were seen in the control versus the experimental. Following a diacetyl rest both beers were tasted by a trained panel. No difference in the taste of the beers was detected by the panel.

Stout Trial A control Stout brew (10.4°F) was pitched with cropped yeast from a previous fermentation. A second brew, the experimental brew, was pitched with the fed batch propagated yeast in a separate fermentation vessel (FV) having the same dimensions as that of the control brew. Both fermentations reached their attenuation limit after 4 days and samples were taken for volatile analysis at the end of fermentation. The results of the volatile analysis are shown in Figure 5. Ester levels were similar in the experimental versus the control. The levels of combined higher alcohols were higher in the control stout brew compared to the experimental brew, 208 ppm versus 148 ppm respectively. The acetaldehyde level was lower in the experimental beer compared to the control (<1.25 ppm versus 9.0 ppm). The level of vicinal diketones in the green beers is shown in more detail in Table 3.

Vicinal Diketone Control Experimental free diacetyl <0.01 0.1 total diacetyl 0.02 0.1 free pentanedione-2,3 <0.01 0.0 total pentanedione-2,3 <0.01 0.03 Table 3. The level of vicinal diketones at the end of fermentation in stouts Although of lesser importance for the ist brew than the other volatiles, as the vicinal diketone levels are generally very well controlled within a brewery, similar levels were seen in the control stout brew versus the experimental brew. Following a diacetyl rest both beers were tasted by a trained panel. No difference in the taste of the beers was detected by the panel.

The process described herein enables yeast to be grown to very high cell densities such that the yeast propagation process can be scaled down compared to known batch yeast propagation processes. Advantageously, the yeast propagation process according to the present teaching can be scaled down such that the entire process can be conducted in a vessel which is small enough to be skid mounted. The skilled person will appreciate that a skid is an apparatus comprising a mobile plant that can be used in the propagation process.

The yeast propagation process described herein produces a higher yeast cell count at the end of propagation compared to known processes. The yeast produced comprises from 1,000 million cells per ml to 2,500 million cells per ml such that the size of the propagation plant can be reduced by a factor of 10 to 25 times compared to a conventional yeast propagation system. This results in significant cost savings for a brewery or distillery. For example, known conventional yeast propagation systems for a brewery can cost of the order of 1.3 million. The scaled down system which can be used for the yeast propagation process according to the present teaching costs significantly less, for example, in the region of 200k.

The present teaching also provides a skid mounted apparatus for use in breweries and distilleries for carrying out the propagation process according to the present invention. As shown in Figure 6, the fermentation skid 100 comprises a jacketed dish-bottomed stirred tank reactor (STR) 101 with CIP (cleaning in place) and SIP (sterilization in place) capability. The STR 101 is equipped with a top-mounted agitation motor 102 with an appropriate mechanical seal. The top-plate assembly may be attached to the vessel using appropriate bolts. The top plate assembly contains a pressure relief valve, a vacuum relief valve, a pressure indicator and ports for insertion of sensors, feed-medium, and CIP liquids. The shaft 108 of the agitator is capable of supporting 3 Rushton 6-blade impellers 109 and is capable of 1200 revolutions per min. Suitable ports for sensors are incorporated into the vessel. A steam sterilizable sample port is incorporated into the vessel. A single port for the insertion of a pH probe is provided at the base of the vessel. An outlet is incorporated at the base of the vessel to facilitate the transfer of the yeast culture.

A second vessel, a fed-batch medium vessel 200, with CIP and SIP capability, is mounted on the skid 100 so that the feed medium can be sterilized in place. A mixer 201 is fitted to this vessel and the agitation motor 202 is mounted on top of this vessel. A pump 203 is provided to control the flow of the feed medium from this second vessel to the STR during the fed-batch phase of the process.

For economic reasons, in-house fed batch fermentations have not found favour with brewers or distillers, especially those who use a single yeast strain and use their propagators sporadically. The advantages of propagating yeast in-house compared to what is normal custom and practice in the brewing and distilling industry include low capital cost and lower operating costs due to lower energy and water requirements. For example, the fed-batch propagation process according to the present teaching produces high quality viable yeast which comprises high yeast cell numbers. This results in a smaller footprint requirement for propagation apparatus for use in carrying out the process described herein. The propagation time for the process described herein is also flexible (variable process time of 24 to 44 hours) as the specific growth rate is a process parameter, compared to inflexible conventional propagation processes which can take up to 48 hours. Furthermore, in conventional yeast propagation processes, blending is generally required to remove off-flavours produced during the process. Advantageously, the process described herein removes off-flavours, such as acetaldehyde, so that when the yeast produced is used to inoculate wort, the beer produced in the beer production process has the correct flavour profile and does not have to be blended off. In addition, in the case of conventional pitching of spirit fermentations, there can be a reduction in the yield of ethanol due to bacterial contamination when cream yeast (bulk) or active dry yeast is used. In contrast, the in-house fed-batch propagation process described herein uses pure yeast culture and there is no loss in ethanol yield due to bacterial contamination of the yeast. The use of the in-house fed-batch propagation system according to the present application therefore redresses the disadvantages associated with active dry yeast while maintaining the advantages associated with fed-batch propagation process described herein.

Yeast propagated in breweries is normally aerated intermittently in wort and generally reaches a cell density of approximately 100 million cells m1-1. This yeast is transferred when it reaches exponential phase, however the beer produced from this 1st generation yeast is usually blended. The yeast crop from the second crop (2nd generation yeast) would however produce beer with a typical flavour. This process not just generates re-work, that is, blending, it is very wasteful in capital expenditure as the yeast propagation plant has be very large to accommodate such low-density propagations. In contrast, the process according to the present teaching produces high density yeast cultures such that the propagation plant can be smaller in size.

In the process according to the present teaching, a very high level of acetaldehyde is produced during the batch phase but this is significantly reduced following the fed-batch phase. Analysis of Figure 3 shows that the acetaldehyde is removed in the absence of either respiration or fermentation as no carbon dioxide is produced during this phase.

The acetaldehyde (and other volatiles such as higher alcohols) is removed by the physical action of the sterile air being supplied to the STR stripping the volatiles from the medium. The results in Figure 3 demonstrate that the stripping action of the air alone will remove substantially all of the acetaldehyde.

It will be appreciated by the person skilled in the art, that not all yeast propagation processes are suitable for the production of yeast for use in the production of beverage alcohol. In particular, the flavour profiles of certain known yeast propagation processes do not produce the correct flavour profile in the main (beer) fermentation. In contrast, the fed-batch process according to the present teaching enables the production of beers having a desirable flavour compound profile. The results demonstrate that the flavour compound profiles of the beers produced using the fed-batch process according to the present teaching show that they are virtually identical from a chemical analysis perspective and identical from a flavour perspective from control beers.

The words comprises/comprising when used in this specification are to specify the presence of stated features, integers, steps or components but does not preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

Claims

Claims 1. A process for the propagation of an alcohol beverage yeast comprising: (i) inoculating a medium with an alcohol beverage yeast strain and initiating a batch phase propagation to generate yeast at a first yeast growth rate within a batch; (ii) maintaining the batch phase propagation until the first yeast growth rate reduces to a second yeast growth rate; (iii) maintaining an aeration of said batch during a period of between 10 and 20 hours from said initiation of said second yeast growth rate; and (iv) subsequently adding a feed medium and conducting a fed-batch phase propagation to generate the alcohol beverage yeast, the fed-batch phase propagation having a third yeast growth rate, the third yeast growth rate being greater than the second yeast growth rate; wherein said fed-batch propagation is conducted at a specific growth rate in the range 0.08 to 0.18 hr' andacetaldehyde levels at the end of the process are less than 37 ppm.
2. A process according to claim 1, wherein said alcohol beverage yeast comprises a high cell density yeast culture comprising from 1,000 to 2,500 million cells per ml.
3. A process according to claim 1 or 2 wherein the medium in step (i) comprises malt extract in an amount 122 to 140 g/L, potassium salt in an amount 2 to 3 g/Land zinc ions in an amount 0.1 to 0.2 ppm Zn2+.
4. A process according to claim 3 wherein the medium comprises malt extract in an amount 132 g/L, potassium dihydrogenphosphate (KH2PO4) in an amount 2.5 g/L and zinc ions in an amount 0.00015 g/L.
5. A process according to any one of claims 1 to 4 wherein the feed medium comprises one or more of the following: malt extract in an amount of 300 to 350 g/L, yeast extract in an amount of 5 to 15 g/L, preferably 10 g/L, and glucose in an amount of 300 to 350 g/L.
6. A process according to any one of claims 1 to 5 wherein acetaldehyde levels at the end of the process are <3 ppm.
7. A process according to any preceding claim further comprising the step of maintaining the pH in the range 4.9 to 5.1, preferably pH 5.
8. A process according to any preceding claim, wherein batch phase propagation is conducted for a period of time in the range 14 to 20 hours.
9. A process according to any preceding claim, wherein the fed batch phase is conducted for a period of time in the range 8 to 24 hours at a specific growth rate in the range 0.1 to 0.17 h-1.
10. A process according to any preceding claim, wherein the Kea during batch phase propagation is in the range from 0.02 to 0.05 S-1, preferably, 0.03 and 0.04 s-1.
11. A process according to any preceding claim, wherein the alcohol beverage yeast strain is selected from the group consisting of ale yeast strain, Lif1, Ale, lager, Weiss bier, stout, whisk(e)y, grain neutral spirit, and yeasts.
12. Yeast propagated according to the process of any one of claims 1 to
13. A method of fermentation for the production of an alcohol beverage comprising use of yeast propagated according to the process of any one of claims 1 to 11.
14. A method according to claim 13, wherein the level of acetaldehyde in the alcoholic beverage is <5ppm, preferably <3 ppm.
15. A method according to claim 14, wherein the level of acetaldehyde in the alcoholic beverage is <1.25ppm.
16. A method according to any one of claims 13 to 15, wherein the alcohol beverage is selected from the group consisting of beer, lager, ale and whisk(e)y.
17. A growth medium for use in propagation of an alcohol beverage yeast; said medium comprising: Malt extract in an amount in the range 122 to 140 g/L; Potassium salt in an amount in the range 2 to 3 g/L; Zinc ions in an amount in the range 0.1 to 0.2 ppm Zn2+g/L; and additional nutrients and/or vitamins.