WO2026003149A1

WO2026003149A1 - Method for producing a hydrated frozen yeast

Info

Publication number: WO2026003149A1
Application number: PCT/EP2025/068026
Authority: WO
Inventors: Philipp Paul GRUENERT
Original assignee: Chr Hansen AS
Current assignee: Chr Hansen AS
Priority date: 2024-06-26
Filing date: 2025-06-26
Publication date: 2026-01-02
Anticipated expiration: 2026-12-26

Abstract

The present invention relates to a method of preparing a hydrated, frozen yeast; a hydrated, frozen yeast obtained through the method; a frozen composition comprising the hydrated, frozen yeast; and use of the frozen composition for producing a fermented beverage.

Description

TITLE METHOD FOR PRODUCING A HYDRATED FROZEN YEAST FIELD The present invention relates to a method of preparing a hydrated, frozen yeast; a hydrated, frozen yeast obtained through the method; a frozen composition comprising the hydrated, frozen yeast; and use of the frozen composition for producing a fermented beverage. This is relevant to the beverage industry in general, and especially to the winemaking and beer brewing industry. BACKGROUND For the last 50 years winemakers have become accustomed to inoculating their grape juice with pure cultures of yeast, also known as "starter cultures". Most commonly, a dehydrated form of yeast called active dried yeast (ADY) is used as a starter culture. To produce ADY, a yeast is propagated in fermenters, concentrated and/or filtered, and then either dried on a drum, or through a process called fluid bed drying. In other instances, the drying is instead carried out by freeze-drying, producing freeze-dried yeast (FDY). It is widely accepted that ADY must be rehydrated before addition to fruit juice. The rehydration is a very delicate process requiring a significant amount of time and attention to ensure that the yeast cells hydrate to a metabolically active and viable state. Incorrectly rehydrating ADY can result in loss of activity or a stuck alcoholic fermentation (O'Kennedy. (2008). How to avoid stuck fermentations, The Australian & New Zealand Grapegrower & Winemaker. November, Issue 538, 103-105; Soubeyrand et al. (2006) Rehydration Protocols for Active Dry Wine Yeasts and the Search for Early Indicators of Yeast Activity). Additionally, the rehydration step increases the number of contamination sources, leading to an overall risk of spoiling a batch. As discussed on page 124, left column of the textbook “Principles and Practices of Winemaking” by Roger Boulton et al, 1996, during the drying process of active dried yeast production, the permeability of the yeast’s plasma membrane increases significantly. To re-establish normal functionality of the plasma membrane, it is important to rehydrate the membranes by adding the yeast to water at 40°C for 20 minutes. Therefore, the ADY rehydration process typically involves a 20-30 minute rehydration step, wherein the ADY is suspended in unchlorinated water or a water / grape juice mix (2:1) between 35-38°C then followed by the addition of grape juice of the same volume (50:50 grape juice/water blend) which is kept for another 20-30 minutes before adding it to the grape juice. It is important that the grape juice should not contain any SO2, which could kill the yeast cells during the sensitive process of rehydration (O'Kennedy. (2008). How to avoid stuck fermentations, The Australian & New Zealand Grapegrower & Winemaker. November, Issue 538, 103- 105). In addition, yeast generally does not tolerate abrupt temperature changes, i.e., thermal shock, even when the temperature changes are within temperatures that, when held constant, would not stress the yeast. Rehydrated yeast should therefore be acclimatized slowly to the grape juice temperature, e.g., by addition of small amounts of the grape juice at the grape juice temperature. O'Kennedy recommends that the changes in temperature should not exceed 5 °C at a time and breaks of 15 minutes should be held between each addition of grape juice. Failure to acclimatize from the rehydration temperature to the grape juice temperature after 30 minutes can also result in significant cell death. Furthermore, care should be taken to use uncontaminated grape juice for the rehydration protocol as rehydration with contaminated grape juice will result in contamination of all wine fermentation inoculated using the rehydration mixture. Different manufacturers propose variations of carrying out the rehydration, but the critical step is that dehydrated cells need to be exposed to water or a water/grape juice mixture at specific temperatures, conditions and for specific time intervals to hydrate properly, thereby avoiding cell death and consequent in-activity. It is not usually recommended to add the ADY directly to the grape juice as the high sugar concentration, SO2 and other compounds in grape juice do not allow for optimal rehydration of the yeast. For this reason, none of the wine yeast manufacturer proposes the direct inoculation of ADY to grape juice. To avoid the drawbacks of having to rehydrate the yeast, the yeast can instead be provided in a hydrated, frozen form. As described in EP2563169B1, such a product is produced by propagating a yeast in a fermenter, concentrating the propagated yeast, and adding a cryoprotectant to the concentrated yeast before freezing it at 50°C in a suitable container. Since the yeast is frozen in a hydrated form, the yeast need not be rehydrated, and may instead, after thawing, be directly added to the grape juice. Importantly, most of the cells are recovered after thawing, with 88.7% of the cells surviving thawing. The survival is made possible by the addition of cryoprotectants, which prevent the formation of sharp ice crystals that burst the yeast cells. Direct inoculation of yeast thawed from a frozen form results in significant time and man-power savings for winemakers as compared to rehydrating ADY. The rehydration process can take up to an hour for a skilled person to carry out, whereas direct inoculation of frozen yeast takes only a few minutes and does not require any special training or skills to perform. Although yeast in a hydrated, frozen form provides several advantages over dried forms, the frozen form necessities the use cryoprotectants, which are generally undesired as they dilute the beverage. Examples of cryoprotectants include sucrose, trehalose, and glycerol. Also, many cryoprotections are not usable in food applications. For instance, sucrose is usually used for standardization purposes in winemaking, and as such additional sucrose is undesired. Freezing a yeast without a cryoprotectant generally results in a yeast with a low viability, meaning that a large portion of the cells are not live and/or metabolically active. For instance, freezing yeast without a cryoprotectant may results in a freezing-thawing survival rate of less than 60%, 40%, or 20%, or the freezing- thawing process may even lead to no colony forming cells. The low viability is less desirable as it increases the amount of frozen yeast that need to be added to a fruit juice fermentation, which dilutes the product, consumes more resources, and increases costs. Thus, there remains a need for a form of yeast which may be used as a starter culture for a wide range of fermented beverages, which form of yeast can be used in a time-efficient manner and without substantial training, and which has a high viability. SUMMARY The inventors of the present invention have found a method to provide a hydrated, frozen yeast, which method comprises steps that conditions a yeast to be freezing- thawing tolerant irrespective of whether the yeast is frozen in presence of cryoprotectants. By “freezing-thawing tolerant” is meant a cell survival rate after freezing and thawing which is greater than what can be achieved with prior art methods that freezes hydrated yeast in a composition substantially free from cryoprotectants. As such, in a first aspect of the present invention, a method for obtaining a frozen yeast is provided, the method comprising the steps of: i) providing a fluid medium, ii) inoculating a yeast in the fluid medium, iii) contacting a zinc source with the fluid medium, iv) adding a fatty acid source to the fluid medium, preferably a polysorbate, wherein steps ii)-iv) can be performed in any order, v) after the steps of i)-iv), allowing the yeast to drive an aerobic process in the fluid medium thereby obtaining a processed composition comprising a conditioned yeast, and vi) freezing the conditioned yeast. Without wishing to be bound by theory, it is believed that aerobic conditions are necessary for the yeast to adopt a phenotype which is freezing-thawing tolerant and, conversely, that extended oxygen limiting steps negatively impact the freezing-thawing survival rate. It is a general issue that the oxygen demand may outgrow the supply of oxygen provided to the fluid medium by the bioreactor. Such a scenario is shown in figure 1, period X and figure 2, periods A and B, where oxygen limited processes are observed for propagations of Pichia kluyveri in a stirred tank bioreactor. Without wishing to be bound by theory, it is believed that the addition of a fatty acid source, preferably a polysorbate, to the fluid medium lowers the oxygen demand, as oxygen is consumed in the synthesis of fatty acids by yeasts. A fatty acid source may for instance be a polysorbate that is taken up and metabolized by the yeast to a fatty acid or it may simply be a fatty acid. A lower oxygen demand in the presence of a fatty acid source is supported by figures 2 and 3, which shows that yeast propagations with media containing polysorbate 80 do not exhibit oxygen limiting steps. Furthermore, by providing a fatty acid source that, it is assumed that a change in lipid composition of the yeast occurs. Where the fatty acid source is a source of a particular subset of fatty acids or essentially is a source for a single fatty acid, the plasma membrane may even be biased towards a phospholipid composition with an increased proportion of the phospholipids which are metabolic products of the provided fatty acids. Without wishing to be bound by theory, it is believed that the changes to the phospholipid composition may increase the flexibility of the plasma membrane such that the yeast is less prone to puncturing by ice crystals formed during a freezing-thawing process. Surprisingly, propagating yeast in a fluid medium with PS80 and a high zinc concentration further enhanced the freezing-thawing rate relative to propagating with a fluid media with PS80, as shown in table 4. The inventors currently do not know why combining higher zinc concentrations with PS80 leads to a synergistic improvement in freezing-thawing survival. It has been shown that Cu,Zn superoxide dismutase is involved in yeast’s response to freezing-thawing stress (Park et al. 1998 Sep 4;273(36):22921-8). Propagating yeast in a fluid medium with PS80 and a low zinc concentration also enhanced the freezing-thawing rate relative to propagating with a fluid media with PS80, as shown in table 4. However, increasing the zinc concentration in the fluid medium does not by itself improve the freezing- thawing survival rate, as demonstrated by figure 1 and figure 2, wherein yeast propagations with 0.006 and 0.012 kg/m³ zinc sulphate monohydrate resulted in comparable survival rates in the range of 60-80%. Without wishing to be bound by theory, it is hypothesized that both an aerobic process and zinc, preferably regardless of the zinc concentration, are essential for an optimal freezing-thawing stress response. Considering the above, the hydrated, frozen yeast obtained through the method of the present invention is clearly advantageous as a starter culture in, for example, winemaking, as the benefits of a hydrated, frozen yeast can be realized without the use of cryoprotectants. I.e., the hydrated, frozen yeast may be thawed and directly inoculated into grape juice without prior rehydration, and the yeast will substantially not add cryoprotectants to the grape juice. The results provided herein have been obtained by using the wine yeast Pichia kluyveri, Saccharomyces cerevisiae (brewer’s yeast), the lager beer yeast Saccharomyces pastorianus, the wine and beer yeast Lachancea thermotolerans, and the low-alcohol beer yeast Saccharomycodes ludwigii. The skilled person may easily transfer the technology to any other yeast, such as Candida fructus, Citeromyces matritensis, Debaromyces hansenii, Hanseniaspora guillermondii, Hanseniaspora osmophila, Hanseniaspora uvarum, Kluyveromyces marxianus, Kluyveromyces thermotolerans, Pichia fermentans, Pichia membranifaciens, Saccharomyces pastorianus, Saccharomyces bayanus, Schizosaccharomyces pombe, Torulaspora delbrueckii, Zygosaccharomyces bisporus, Zygosaccharomyces rouxii, etc. The yeast obtained through the method of the present invention could also be applied to produce other beverages than wine such as beer, cider, sake, kefir, soft drinks, coffee, juice, tea, and beverages where the action of the particular yeast is required and applied in a convenient, direct inoculation form. The starter culture may be used to ferment any type of sugar based medium, such as grape juice, or apple juice, or alternatively, whole fruits. DEFINITIONS Zn and zinc are synonyms and interchangeable and refers to the chemical element zinc. Polysorbates are a class of emulsifiers obtained by esterification of ethoxylated sorbitan with fatty acids. Non-limiting examples are polyoxyethylene (20) sorbitan monolaurate (also known as polysorbate 20 or PS20), polyoxyethylene (20) sorbitan monopalmitate (also known as polysorbate 40 or PS40), polyoxyethylene (20) sorbitan monostearate (also known as polysorbate 60 or PS60), polyoxyethylene (20) sorbitan tristearate (also known as polysorbate 65 or PS65), and polyoxyethylene (20) sorbitan monooleate (also known as polysorbate 80 or PS80). The use of “conditioned” as in “conditioned yeast” should be understood as a yeast, which was obtained from at least the methods steps i)-v). In the context of the present disclosure, a cryoprotectant should be understood as any substance which prevents damage to cells during freezing. Cryoprotectant are known to work through mechanisms such as interactions with lipid membranes, stabilization of proteins during freezing–thawing processes, and inhibition of formation of intracellular ice. In the context of the present disclosure “substantially free from cryoprotectants” should be understood as less than 0.5wt% of cryoprotectants, such as less than 0.25wt%, such as less than 0.1wt%, such as less than 0.05wt%, such as less than 0.01wt%. In the context of the present disclosure, a cryoprotectant is different from zinc or polysorbate or fatty acid. Preferably, in the context of the present disclosure, a cryoprotectant is mannitol, sorbitol, sodium tripolyphosphate, xylitol, glycerol, raffinose, maltodextrin, erythritol, threitol, trehalose, glucose, fructose, dimethyl sulfoxide (DMSO), ethylene glycol, propylene glycol, or mixtures thereof. In the context of the present disclosure, fatty acid source means any compound that comprises a fatty acid or is a fatty acid, which compound may be taken up and metabolized by the yeast to, for example, phospholipids. In the context of the present disclosure, an aerobic process is a process in which the yeast metabolizes a nutrient through aerobic respiration. In particular, an aerobic process may be defined as a process carried out in the presence of oxygen and in which oxygen is used to metabolize a nutrient such as glucose to carbon dioxide and water, biomass and ATP. In some aspects of the invention, an aerobic process may be a respiro-fermentative process known as Crabtree-Effect were a shift from respiration to fermentation occurs even if oxygen is available. In this later process, ethanol production may vary but is generally minimized.In the context of the present disclosure, an oxygen limited process or step should be understood as a process wherein the rate of yeast biomass accumulation decreases during a period of maximal oxygen supply to the fluid medium. By oxygen supply is meant the oxygen provided to the fluid medium by action of a bioreactor used for propagating the yeast. The maximal oxygen supply thus depends on the bioreactor used. Mechanism to provide oxygen to the fluid medium are well-known by the skilled person, e.g., agitation or blowing air into the fluid medium through a sparger, in particular this includes aeration with pure oxygen or oxygen enrichment/pulsed injection into the fermentation device. In the context of the present disclosure, an oxygen limited process is further defined by the period of maximal oxygen supply being for at least 15 min, such as at least 30 minutes, such as at least 60 minutes. In the context of the present disclosure, substantially no fatty acid sources should be understood as below 0.05wt% fatty acid sources. The skilled person would in the present context understand that when you thaw a hydrated, frozen yeast, and inoculate the thawed yeast into a fruit juice, there is no rehydration of the yeast, as the yeast retains its hydrated state also after thawing. BRIEF DESCRIPTION OF FIGURES Figures 1-9 show line graphs of dissolved oxygen (DO) and agitation over time for propagations of different yeasts in a stirred tank bioreactor (STR) using fluid media with or without polysorbate 80 and with different zinc levels. Also shown on the graphs are freezing-thawing survival rates of the yeasts at different harvest time points, the harvest time points being indicated with solid vertical lines. Below, the yeast, zinc content, and polysorbate 80 content is indicated for each figure. Figure 1, Pichia kluyveri propagation with a fluid medium containing 0.006 kg/m³ zinc sulphate monohydrate and substantially no fatty acid sources. The figure also shows a period of oxygen limitation is indicated with brace X. Figure 2, Pichia kluyveri propagation with a fluid medium containing 0.012 kg/m³ zinc sulphate monohydrate and contains substantially no fatty acid sources. Periods with oxygen limitation are indicated with braces A and B. Figure 3, Pichia kluyveri propagation (aerobic) with a fluid medium containing polysorbate 80 and 0.006 kg/m³ zinc sulphate monohydrate. Figure 4, Pichia kluyveri propagation (aerobic) with a fluid medium containing polysorbate 80 and 0.012 kg/m³ zinc sulphate monohydrate. Figure 5, Saccharomyces cerevisiae propagation (aerobic) with a fluid medium containing polysorbate 80 and approx. 0.018 kg/m³ zinc sulphate monohydrate. Figure 6, Saccharomyces pastorianus propagation (aerobic) with a fluid medium containing polysorbate 80 and approx. 0.018 kg/m³ zinc sulphate monohydrate. Figure 7 Lachancea thermotolerans propagation (aerobic) with a fluid medium containing polysorbate 80 and approx. 0.012 kg/m³ zinc sulphate monohydrate. Figure 8, Saccharomycodes ludwigii propagation (aerobic) with a fluid medium containing approx. 0.018 kg/m³ zinc sulphate monohydrate and substantially no fatty acid sources. Figure 9, Saccharomycodes ludwigii propagation (aerobic) with a fluid medium containing polysorbate 80 and approx. 0.018 kg/m³ zinc sulphate monohydrate. DETAILED DESCRIPTION In an embodiment, in step vi) the conditioned yeast is frozen by freezing the processed composition. In an embodiment, the method according to the invention further comprises the step of: - before the step of vi) concentrating the processed composition to obtain a concentrated composition, and wherein in step vi) the conditioned yeast is frozen by freezing the concentrated composition. In an embodiment, the method according to the invention further comprises the steps of: a) providing an auxiliary fluid medium, b) obtaining an auxiliary composition either by b1) isolating at least a portion of the conditioned yeast from the processed composition, and b2) mixing the isolated conditioned yeast obtained in step 1a) with the auxiliary fluid medium to obtain to the auxiliary composition, or by b3) concentrating at least a portion of the processed composition to obtain a concentrated composition, and b4) mixing at least a portion of the concentrated composition with the auxiliary fluid medium to obtain the auxiliary composition, and wherein in step vi) the conditioned yeast is frozen by freezing the auxiliary composition. In an embodiment, the method according to the invention does not comprise an oxygen limited process or an oxygen limited step. In an embodiment, the method according to the invention does not comprise a step of reducing the moisture content of the yeast. The fatty acid source may be any compound that comprises a fatty acid or is a fatty acid, which compound may be taken up and metabolized by the yeast to, for example, a phospholipid. A compound that comprises a fatty acid could for instance be a compound, which comprises a fatty acid ester, such as a polysorbate. The fatty acid may be any fatty acid. In a specific version of this embodiment, the fatty acid is an unsaturated fatty acid, such as a monounsaturated fatty acid or a polyunsaturated fatty acid. In an embodiment the fatty acid source is water miscible. In another embodiment the fatty acid source is an emulsifier. In an embodiment, the fatty acid source is a polysorbate, such as polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, or polysorbate 80. In a preferred version of this embodiment, the fatty acid source is polysorbate 80. In an embodiment, the fatty acid source is an unsaturated fatty acid source, such as polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, or polysorbate 80. In a preferred version of this embodiment, the fatty acid source is polysorbate 80. In a preferred embodiment, the polysorbate is added or may be added to the fluid medium in a concentration of 0.20-20 kg/m³, preferably 0.5-15 kg/m³, more preferably 1.0-10 kg/m³, even more preferably 1.5-5 kg/m³. Foam is or may be generated with 25 kg/m³ or more of polysorbate. In a preferred embodiment, polysorbate 20 or polysorbate 40 or polysorbate 60 or polysorbate 65 or polysorbate 80 is added or may be added to the fluid medium in a concentration of 0.20-20 kg/m³, preferably 0.5-15 kg/m³, more preferably 1.0- 10 kg/m³, even more preferably 1.5-5 kg/m³. The zinc source may be any material that provides Zn²⁺ to the fluid medium. By “contacting a zinc source with the fluid medium” is meant either contacting a material with the fluid medium, which material releases Zn²⁺ to the medium, or the addition of a zinc source to the fluid medium. For example, Zn²⁺ may be provided to the fluid medium by e.g., a zinc or zinc alloy surface of a bioreactor which is in contact with the fluid medium. A surface of the bioreactor could be an agitator or a container of the bioreactor. Examples of addition of zinc sources to the fluid medium could be the addition of zinc sulphate monohydrate to the fluid medium. Other examples are zinc chloride, zinc acetate, zinc carbonate, etc. Another example could be the addition of an organic substance containing zinc, such as dead/inactive yeast or yeast extracts. A dead yeast may for instance be a zinc-enriched yeast. In an embodiment, in the step of iii) metallic zinc or an alloy comprising zinc is contacted with the fluid medium. In a specific version of this embodiment, the metallic zinc or the alloy comprising zinc allows for at least 2.5 millimoles of zinc per kg fluid per hour. In an embodiment, in the step of iii), a zinc source providing at least 50 millimoles of zinc per kg fluid medium is added to the fluid medium, such as at least 55 millimoles zinc per kg fluid medium, such as at least 60 millimoles zinc per kg fluid medium, such as at least 65 millimoles zinc per kg fluid medium, such as at least 66 millimoles zinc per kg fluid medium. In an embodiment, in the step of iii) zinc is contacted with the fluid medium by adding a zinc salt to the fluid medium. In a specific version of this embodiment, the zinc salt is added in an amount provide least 50 millimoles zinc per kg fluid medium, such as at least 55 millimoles zinc per kg fluid medium, such as at least 60 millimoles zinc per kg fluid medium, such as at least 65 millimoles zinc per kg fluid medium, such as at least 66 millimoles zinc per kg fluid medium. In a specific version of this embodiment, the zinc salt is a zinc sulphate, preferably zinc sulphate monohydrate. In an embodiment, in the step of iii) the zinc or zinc source or zinc salt is contacted with the fluid medium in a concentration 10-250 millimoles zinc per cubic meter fluid medium, preferably 30-150 millimoles zinc per cubic meter fluid medium, more preferably 40-125 millimoles zinc per cubic meter fluid medium, even more preferably 50-100 millimoles zinc per cubic meter fluid medium. It is known to the skilled person how to calculate the number of moles of zinc to add to the method regardless of the source of zinc used, for example zinc sulphate monohydrate versus zinc sulphate heptahydrate. In a specific version of this embodiment, the zinc salt is a zinc sulphate, preferably zinc sulphate monohydrate. The fluid medium may be any medium suitable for propagating a yeast. The medium may, e.g., be a defined or complex medium or media in a batch or fed-batch fermentation set-up as known to the skilled person but may as well be applicable to derivative fermentation technologies such as perfusion, semi-continuous culture (also known as repeated fed-batch) or even continuous fermentation when harvested from the outflow that is consequentially similar to end of fermentation of a batch or fed-batch set-up. Such media typically comprise a buffer component (such as a phosphate, sulphate or citrate buffer, or mixtures thereof), an antifoam agent or antifoam agent mixture (e.g., silicon oil, polyether-based antifoam agent or natural oil antifoam agent, i.e., hops-based antifoam agent), sources of amino acids, sources of small peptides, and sources of vitamins (such as steep liquor, yeast extract, soy, pea, or similar plant-derived materials), organic or inorganic nitrogen sources (such as ammonium sulphate, ammonium chloride, amino acids, etc.), trace elements and metals (such as salts of potassium, calcium, iron, magnesium, copper, zinc, manganese, iodine, sodium, molybdian, cobalt, and boron), a pure or a mixed carbon source (such as glucose, glucose syrup, dextrins, maltodextrins, sucrose, maltose, fructose, glucose-fructose syrups, fructose- syrups, maltose syrups, malt extracts, glycerol, maltotriose, maltotetraose, trehalose, molasses, high fructose corn syrup, thick juice, and similar). The skilled person knows how to formulate medium for propagation of different yeasts, different reactor volumes, different bioreactor types, and combinations thereof. In an embodiment, the yeast is selected from Pichia kluyveri, Saccharomyces cerevisiae, Saccharomyces pastorianus, Lachancea thermotolerans, Saccharomycodes ludwigii, Candida fructus, Citeromyces matritensis, Debaromyces hansenii, Hanseniaspora guillermondii, Hanseniaspora osmophila, Hanseniaspora uvarum, Kluyveromyces marxianus, Kluyveromyces thermotolerans, Pichia fermentans, Pichia membranifaciens, Saccharomyces pastorianus, Saccharomyces bayanus, Schizosaccharomyces pombe, Torulaspora delbreuckii, Torulaspora delbrueckii, Zygosaccharomyces bisporus, and Zygosaccharomyces rouxii. Preferably, the yeast is selected from Pichia kluyveri, Saccharomyces cerevisiae, Saccharomyces pastorianus, Saccharomycodes ludwigii, Lachancea thermotolerans, and Torulaspora delbrueckii. In an embodiment, the processed composition is not a matrix. In another embodiment, the concentrated composition is not a matrix. In yet another embodiment, the auxiliary composition is not a matrix. A matrix may be any composition which comprises a two-phase system that consists of a liquid phase and a solid phase, the solid phase being built from a network of molecules. Examples of matrices are a dough, a gel, etc. A matrix can be a should not be confused with a dispersion. In the context of the present application, a dispersion is a heterogeneous mixture of a fluid and cells and/or particles. I.e., if cells and/or particles are dispersed in a liquid medium do not form a network, they cannot constitute a matrix and are instead considered a dispersion. In an embodiment, the processed composition is a dispersion. In another embodiment, the concentrated composition is a dispersion. In yet another embodiment, the auxiliary composition is a dispersion. In an embodiment, the processed composition is not a dough. In another embodiment, the concentrated composition is not a dough. In yet another embodiment, the auxiliary composition is not a dough. In an embodiment, the yeast composition has a water content of at least 50wt%, preferably at least 60wt%, even more preferably at least 70wt%, most preferably at least 80wt%. In an embodiment, the processed composition is substantially free from a cryoprotectant. In another embodiment, the concentrated composition is substantially free from a cryoprotectant. In yet another embodiment, the auxiliary composition is substantially free from a cryoprotectant. To be substantially free from a cryoprotectant means that the cryoprotectant is different from zinc, from a polysorbate and/or from a fatty acid. Preferably it means that the cryoprotectant is selected from mannitol, sorbitol, sodium tripolyphosphate, xylitol, glycerol, raffinose, maltodextrin, erythritol, threitol, trehalose, glucose, fructose, dimethyl sulfoxide (DMSO), ethylene glycol, propylene glycol, or mixtures thereof. In a second aspect, the present disclosure provides a frozen yeast obtained through a method according to the first aspect of the present disclosure. In a third aspect, the present disclosure provides a frozen composition comprising a frozen yeast, said frozen yeast being the frozen yeast of the second aspect of the present disclosure. In a fourth aspect, the present disclosure provides a use of a frozen composition for producing a fermented beverage, the frozen composition being according to the third aspect of the present disclosure. EXAMPLES Example 1 Media were prepared by mixing and/or dissolving in water the ingredients as set out in table 1: Table 1 Medium ingredient Amount per volume medium Yeast extract (liquid 50wt%), provided by 20 or 40 kg/m³ Ohly Dextrose monohydrate 0 or 35.00 kg/m³ Dextrose-Dextrin mixture 0 or 85 kg/m³ Potassium dihydrogen phosphate 1.00 kg/m³ Magnesium sulphate heptahydrate 1.00 kg/m³ Antifoam oil (STRUKTOL^® SB 509, containing silicone polymer, non-ionic tensides and 2 or 3.67 L/m³ silica) Ammonium sulphate 2.00 kg/m³ Zinc sulphate monohydrate 0.006 or 0.012 kg/m³ or 0.018 Polysorbate 80 0, 1, or 1.5 kg/m³ Nine propagations were carried out with the following yeasts and media: Table 2 Number Yeast Dextrose Polysorbate Zinc sulphate monohydrate or 80 monohydrate dextrin- (H)\1000kg dextrose (kg/m³) 1 Pichia kluyveri Dextrose Low (0.006) Without (0) monohydrate 2 Pichia Dextrose High (0.012) Without (0) 3 Pichia kluyveri Dextrose Low (0.006) With (1.0) monohydrate 4 Pichia kluyveri Dextrose High (0.012) With (1.0) monohydrate 5 Saccharomyces Dextrin- cerevisiae dextrose mix 6 Saccharomyces Dextrin- High (0.018) With (1.5) pastorianus dextrose mix 7 Lachancea Dextrose High (0.012) With (1.0) thermotolerans monohydrate 8 Saccharomycodes Dextrose High (0.018) Without (0) ludwigii monohydrate 9 Saccharomycodes Dextrose High (0.018) With (1.5) ludwigii monohydrate Propagation of yeast in each of the media were carried out either using a 30 L technical or 2 L lab-scale stirred tank bioreactor (STR) equipped with a dissolved oxygen (DO) sensor. Propagations of Pichia kluyveri were carried out over 20-40 hours depending on media composition at 30°C with DO-regulated agitation set to 20% DO (10% DO in the case of low zinc and without PS80). Propagation of Saccharomyces cerevisiae was carried out over 65-70 hours at 25°C with DO- regulated agitation set to 20% DO. Propagation of Saccharomyces pastorianus were carried out over 52-55 hours at 25°C with DO-regulated agitation set to 30% DO. Propagation of Lachancea thermotolerans were carried out over 47-50 hours at 25°C with DO-regulated agitation set to 20% DO. Propagation of Saccharomycodes ludwigii were carried out over 27-31 hours at 25°C with DO-regulated agitation set to 30% DO (20% DO in the case without PS80). For all propagations, agitation (rpm) and DO was recorded throughout the propagation at a sampling frequency of 0,016 Hz/1,0 min^-1 and a Proportional – Integral (PI) controller was used for the DO-regulated agitation. Yeasts were harvested after propagation at different time points, as shown in table 3: Number Time point 1 Time point 2 Time point 3 1 24 h 2 18 h 20 h 22 h 3 16.5 h 18 h 22 h 4 17 h 19 h 21 h 5 54.4 h - - 6 71.0 h - - 7 47.75 h - - 8 30.5 h - - 9 30 h - - Each of the harvested yeasts were centrifugated to packed cell volumes (PCV) values suitable for production scenarios. The centrifugated yeast were then isolated and frozen by cooling at a rate of less than 1 K/minute to -50°C. The frozen yeasts were then kept at -45°C to -50°C for one week before being thawed for analysis. Cell counts after centrifugation but before freezing and cell counts after freezing and thawing were performed. Cell counts were performed as colony forming units plate counts on YGC agar plates as described in “Compendium of international methods of analysis – OIV – Microbial analysis of wines and musts”, OIV-MA-AS4- 01 : R2010; 6.1. Freezing-thawing survival rates were calculated as the number of cells after freezing and thawing divided by the number of cells before freezing and thawing. The freezing-thawing survival rates are summarized in table 4: Freezing-thawing survival rate Yeast Propagated Propagated Propagated Propagated with low with high with low with high zinc, no zinc, no zinc, and zinc, PS80 PS80 PS80 and PS80 Pichia kluyveri 60-80% 69-74% 74-92% 85-115% Saccharomyces - - - 100% cerevisiae Saccharomyces - - - 95.2% pastorianus Lachancea - - - 100% thermotolerans Saccharomycodes - - 25.4% 69% ludwigii As can be seen from table 4, increasing the zinc concentration from 0.006 to 0.012 kg/m³ zinc sulphate monohydrate alone did not affect the freezing-thawing survival. Adding PS80 at 1 kg/m³ to a media with low zinc (0.006 kg/m³ zinc sulphate monohydrate) improved the freezing-thawing survival rate significantly and surprisingly relative to the equivalent media with low zinc, from 60-80% to 74- 92%. This clearly shows a synergistic effect of combining low zinc levels with PS80. Surprisingly, yeast which were propagated in a medium comprising both PS80 at 1- 1.5 kg/m³ and high zinc (0.012 or 0.018 kg/m³ zinc sulphate monohydrate) had an even higher freezing-thawing survival rate compared to the equivalent media without PS80. This clearly shows a synergistic effect of combining high zinc levels with PS80. Figures 1-9 display the recorded agitation and DO over time, as well as the freezing-thawing survival rates for propagations 1-9, respectively. Data was Savitzky–Golay (SG) filtered (smoothed) over 25 datapoints for readability. Figure 1 shows the DO and agitation over time for a propagation of Pichia kluyveri using a medium with a low zinc concentration of 0.006 g/L zinc sulphate monohydrate without the addition of polysorbate 80 (or any other fatty acid source). The agitation increases according to the rising volumetric oxygen demand due to the proliferating yeast and hits the maximum agitation of the STR resulting in DO limitation in period X. Figure 2 shows the DO and agitation over time for a propagation of Pichia kluyveri using a medium with an increased (high) zinc concentration of 0.012 g/L zinc sulphate monohydrate without the addition of polysorbate 80 (or any other fatty acid source). The overall appearance of the propagation trajectories did not change compared to the one seen in figure 1. However, an increase in biomass yields was observed (data not shown). The starting DO appear elevated due to an increased head space pressure set to minimize risk of contaminants entering the fermenter; this is a common standard measure in propagation. The agitation increases according to the rising volumetric oxygen demand due to the proliferating yeast and hits the maximum agitation of the STR twice resulting in DO limitation in periods A and B. Without wishing to be bound by theory, it is believed that a diauxic shift from the primary (glucose) to secondary carbon sources (thought to be free amino acids) occurs in between the two phases of DO limitation, resulting in a short period of time of lowered oxygen demand due to adaptation to the new main carbon source. This is believed to have caused the agitation to suddenly drop and the DO to increase accordingly. It was observed that oxygen depletion increased relative to low zinc concentration (0,006 g/L zinc sulphate monohydrate, figure 1), which may be a result of the increased biomass yield. The freezing-thawing survival rate did not change in comparison to the propagation with low zinc concentration, with the survival rate being in the range of 69-74% for high zinc and in the range of 60-80% for low zinc. Figure 3 shows the DO and agitation for a propagation of Pichia kluyveri using a medium with a low zinc concentration of 0.006 g/L zinc sulphate monohydrate and 1 g/L polysorbate 80. The starting DO appear elevated due to the increase head space pressure set to minimize risk of contaminants entering the fermenter. The agitator increases according to the rising volumetric oxygen demand due to the proliferating yeast. Following a plausible diauxic shift from the primary to the secondary carbon sources, short periods of time with a lowered oxygen demand due to adaptation to the new main carbon source caused the agitation to suddenly drop. Only for brief moments did the DO dip below the setpoint when the proliferating culture completely adjusted for the next nutrient. As such, no oxygen limiting steps were observed. Figure 4 shows the DO and agitation for a propagation of Pichia kluyveri using a medium with a high zinc concentration of 0,012 g/L zinc sulphate monohydrate and 1 g/L polysorbate 80. The starting DO appear elevated due to the increase head space pressure set to minimize risk of contaminants entering the STR. The agitator increases according to the rising volumetric oxygen demand of the yeast. Following a plausible diauxic shifts from the primary to secondary carbon sources short periods of time with a lowered oxygen demand due to adaptation to the new main carbon source caused the agitation to suddenly drop whilst the DO was successfully kept at the desired setpoint for this species. It was observed that by combining polysorbate and increasing the zinc concentration, oxygen depletion can be remedied whilst increasing biomass yields. Figure 5 shows a propagation of Saccharomyces cerevisiae using a high zinc concentration of 0.018 g/L zinc sulphate monohydrate and 1.5 g/L polysorbate 80. It appears that oxygen depletion is no issue here as well. This shows that the effect of PS80 is transferable between yeast species. Figure 6 shows a propagation of Saccharomyces pastorianus using a high zinc concentration of 0.018 g/L zinc sulphate monohydrate and 1.5 g/L polysorbate 80. Oxygen depletion is no issue here as well, like the other propagations with polysorbate. This again shows that the effect of PS80 is transferable between yeast species. Figure 7 shows a propagation of Lachancea thermotolerans using a high zinc concentration of 0.012 g/L zinc sulphate monohydrate and 1.0 g/L polysorbate 80. Oxygen depletion is no issue here as well, like the other propagations with polysorbate. This again shows that the effect of PS80 is transferable between yeast species. This was done in 2 L scale where oxygen transfer is quite effective. Figure 8 shows a propagation of Saccharomycodes ludwigii using a high zinc concentration of 0.020 g/L zinc sulphate monohydrate and no polysorbate 80. It is clear that oxygen depletion is no issue here as well. However, the poor freezing- thawing survival rate of only 25.4% highlights that a process without oxygen limiting steps is not enough to replace the effect of PS80, i.e., that likely the improved freezing-thawing resistance of PS80 cannot alone be explained by its oxygen demand lowering effect. Figure 9 shows a propagation of Saccharomycodes ludwigii using a high zinc concentration of 0.020 g/L zinc sulphate monohydrate and 1.0 g/L polysorbate. When comparing with figure 8, we can see a significant improvement in the freezing-thawing survival rate from 25.4% to approx. 69 % with the use of PS80. Again, this highlights the transferability of using zinc and PS80 with different yeasts. Example 2 Effect of zinc and polysorbate (40, 60, 80) and effect of zinc and fatty acids (C18- and C20-), both on propagation of Pichia kluyveri and freezing-thawing survival rate Example 2 was conducted in a manner like Example 1. Example 1 demonstrates and highlights the transferability of using zinc and polysorbate PS80 with different yeasts. Therefore, in Example 2, Pichia kluyveri was chosen as the model yeast to investigate two aspects: the effect of using polysorbates other than polysorbate 80, and the effect of using fatty acids as an alternative to polysorbates. The details of this example are provided below. Table 5 Medium ingredient Amount per volume medium Yeast extract (dry), provided by Ohly 20 kg/m³ Dextrose monohydrate 35.00 kg/m³ Potassium dihydrogen phosphate 1.00 kg/m³ Magnesium sulphate heptahydrate 1.00 kg/m³ Antifoam oil (STRUKTOL^® SB 509, containing silicone polymer, non-ionic tensides and 3.67 L/m³ silica) Ammonium sulphate 2.00 kg/m³ Zinc sulphate monohydrate 0.012 kg/m³ Polysorbate 40, 60, 80 1.0 kg/m³ Oleic acid or arichidic acid 1.1 kg/m³ The following propagations were carried out for Pichia kluyveri: Table 6 Number Zinc sulphate Polysorbate (H)\1000kg (kg/m³) or monohydrate Fatty acid (kg/m³) (kg/m³) 1 0.012 Without polysorbate or fatty acid (0) 2 0.012 With PS40 (1.0) 3 0.012 With PS60 (1.0) 4 0.012 With PS80 (1.0) 6 0.012 With Oleic Acid (1.1) 7 0.012 With Arichidic Acid (1.1) Propagation of yeast in each of the media was carried out using a 2 L lab-scale stirred tank bioreactor (STR) equipped with a dissolved oxygen (DO) sensor. Propagations of Pichia kluyveri were carried out over 20-40 hours depending on media composition at 30°C with DO-regulated agitation set to 20% DO (10% DO in the case of zinc without a polysorbate or fatty acids). For all propagations, agitation (rpm) and DO was recorded throughout the propagation at a sampling frequency of 0,016 Hz/1,0 min^-1 and a Proportional – Integral (PI) controller was used for the DO-regulated agitation. Yeasts were harvested after propagation as follows: harvest was realized manually by transferring the fermentate into a centrifugation container, followed by centrifugation in a desk centrifuge at 4000×g for 15 min. The supernatant was then removed to the extend needed per strain to achieve the desired biomass concentration and liquid contents. Biomass was homogenized manually by stirring. This was done in a way to achieve the same range of biomass content as in production where industrial centrifuges are used either discontinuously or continuously. In a production scenario, the concentrate would be agitated in a concentration tank from which is then filled. Each of the harvested yeasts were centrifugated to packed cell volume (PCV) values suitable for production scenarios. The centrifugated yeast were then filled into tubes and frozen by cooling at a rate of less than 1 K/minute at –80°C for subsequent evaluation purposes. The frozen yeasts were then kept at –80°C for one week before being thawed for analysis. Cell counts were performed on harvested fermentates prior and after centrifugation (then called concentrates), before and after freezing and thawing were performed to evaluate freeze stability (recovery rates). Cell counting was performed using a spread plating methodology determining colony forming units on YGC agar plates as described in “Compendium of international methods of analysis – OIV – Microbial analysis of wines and musts”, OIV-MA-AS4-01 : R2010; 6.1. and using haeomocytometer cell counting chambers to evaluate viability. Freezing-thawing survival rates were calculated as percentage of cells recovered after freezing and thawing in reference to the number of cells before freezing and thawing (plating). In case of haemocytometer determinations the same cell suspensions used to prepare the YGC agar plates were mixed 1:1 with a methyline blue working concentration solution, pipetted into the prepared haemocytometer cell counting chamber and total cell counts as well as blue stained cells were recorded and evaluated microscopically. Freezing-thawing survival rates were calculated based on the viabilities and cell counts determined balancing frozen-thawed against fresh yeast. The freezing-thawing survival rates presented in Table 7. Propagations are made for Pichia kluyveri with 0.012 kg/m³ of zinc sulphate monohydrate. Table 7 Freezing-thawing survival rate No PS, no PS40 PS60 PS80 Oleic Acid Archidic fatty acid Acid 69-82% 86-98% 86-94% 90-100% 41-57% 15-43% Table 7 corroborates the synergistic effect observed in Example 1 for zinc and polysorbate 80, establishing it as a positive control in Example 2. Furthermore, Table 7 demonstrates that the synergistic effect of zinc and polysorbate 80 extends to polysorbate 40 and polysorbate 60, both of which exhibit an increased freezing- thawing survival rate when compared with the negative control (no polysorbate, no fatty acid) and a similar freezing-thawing survival rate when compared with the one obtained for polysorbate 80. Finally, Table 7 supports the choice of using a polysorbate as a preferred approach compared to selecting a fatty acid, such as oleic acid or arachidic acid. Yeast grown with fatty acids shows significantly lower freezing-thawing survival rates compared to yeast grown either without polysorbates or in the presence of polysorbates (40, 60, or 80).

Claims

CLAIMS 1. A method for obtaining a frozen yeast, the method comprising the steps of: i) providing a fluid medium, ii) inoculating a yeast in the fluid medium, iii) contacting a zinc source with the fluid medium, iv) adding a polysorbate to the fluid medium, wherein steps ii)-iv) can be performed in any order, v) after the steps of i)-iv), allowing the yeast to drive an aerobic process in the fluid medium thereby obtaining a processed composition comprising a conditioned yeast, and vi) freezing the conditioned yeast.

2. The method of claim 1, wherein in step vi) the conditioned yeast is frozen by freezing the processed composition.

3. The method of claim 1, the method further comprising the step of: - before the step of vi) concentrating the processed composition to obtain a concentrated composition, and wherein in step vi) the conditioned yeast is frozen by freezing the concentrated composition.

4. The method of claim 1, the method further comprising the steps of: a) providing an auxiliary fluid medium, b) obtaining an auxiliary composition either by b1) isolating at least a portion of the conditioned yeast from the processed composition, and b2) mixing the isolated conditioned yeast obtained in step b1) with the auxiliary fluid medium to obtain to the auxiliary composition, or by b3) concentrating at least a portion of the processed composition to obtain a concentrated composition, and b4) mixing at least a portion of the concentrated composition with the auxiliary fluid medium to obtain the auxiliary composition, and wherein in step vi) the conditioned yeast is frozen by freezing the auxiliary composition.

5. The method according to claims 1 or 2, wherein the processed composition is substantially free from a cryoprotectant, preferably wherein the cryoprotectant is different from zinc, a polysorbate and/or a fatty acid, more preferably wherein the cryoprotectant is selected from mannitol, sorbitol, sodium tripolyphosphate, xylitol, glycerol, raffinose, maltodextrin, erythritol, threitol, trehalose, glucose, fructose, dimethyl sulfoxide (DMSO), ethylene glycol, propylene glycol, or mixtures thereof.

6. The method according to claim 3, wherein the concentrated composition is substantially free from a cryoprotectant, preferably wherein the cryoprotectant is different from zinc, a polysorbate and/or a fatty acid, more preferably wherein the cryoprotectant is selected from mannitol, sorbitol, sodium tripolyphosphate, xylitol, glycerol, raffinose, maltodextrin, erythritol, threitol, trehalose, glucose, fructose, dimethyl sulfoxide (DMSO), ethylene glycol, propylene glycol, or mixtures thereof.

7. The method according to claim 4, wherein the auxiliary composition is substantially free from a cryoprotectant, preferably wherein the cryoprotectant is different from zinc, a polysorbate and/or a fatty acid, more preferably wherein the cryoprotectant is selected from mannitol, sorbitol, sodium tripolyphosphate, xylitol, glycerol, raffinose, maltodextrin, erythritol, threitol, trehalose, glucose, fructose, dimethyl sulfoxide (DMSO), ethylene glycol, propylene glycol, or mixtures thereof.

8. The method according to any one of the preceding claims wherein the method does not comprise an oxygen limited step and/or wherein the method does not comprise a step of reducing the moisture content of the yeast and/or wherein the method does not comprise a step of reducing the moisture content of the yeast.

9. The method according to any one of the preceding claims, wherein the polysorbate is added to the fluid medium in a concentration of 0.20-20 kg/m³, preferably 0.5-15 kg/m³, more preferably 1.0-10 kg/m³, even more preferably 1.5-5 kg/m³.

10. The method according to any of the preceding claims, wherein the polysorbate is selected from polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65, and polysorbate 80, preferably the polysorbate is selected from polysorbate 40, polysorbate 60, and polysorbate 80.

11. The method according to any one of the preceding claims, wherein the yeast is selected from Pichia kluyveri, Saccharomyces cerevisiae, Saccharomyces pastorianus, Saccharomycodes ludwigii, Lachancea thermotolerans, and Torulaspora delbrueckii.

12. The method according to any one of the preceding claims, wherein the method is a batch process or a fed-batch process.

13. The method according to any one of the preceding claims, wherein the zinc source is contacted with the fluid medium in a concentration 10-250 millimoles zinc per cubic meter fluid medium, preferably 30-150 millimoles zinc per cubic meter fluid medium, more preferably 40-125 millimoles zinc per cubic meter fluid medium, even more preferably 50-100 millimoles zinc per cubic meter fluid medium.

14. The method according to any one of the preceding claims, wherein the zinc source is selected zinc chloride, zinc oxide, zinc sulfate, zinc sulfide, zinc acetate, and zinc carbonate.

15. A frozen yeast obtainable by a method according to any one of the claims 1- 14.

16. A frozen composition comprising the frozen yeast of claim 15.

17. Use of the frozen composition according to claim 14 for producing a fermented beverage.