WO2025011924A1

WO2025011924A1 - Freeze-dried composition comprising non-saccharomyces yeast

Info

Publication number: WO2025011924A1
Application number: PCT/EP2024/067500
Authority: WO
Inventors: Hans Bisgaard-Frantzen; Philipp Paul GRUENERT; Sebastian Pohl; Emil Eriksson; Duncan HAMM
Original assignee: Chr Hansen AS
Current assignee: Chr Hansen AS
Priority date: 2023-07-07
Filing date: 2024-06-21
Publication date: 2025-01-16
Anticipated expiration: 2026-01-07

Abstract

The present application is in the field of dry compositions for non-Saccharomyces yeasts, a process for preparing such compositions through freeze-drying and compositions which may be prepared by the processes. The processes are characterized in that the lyoprotectants comprise maltodextrin, antioxidant(s) and disaccharide(s), and that the dry matter weight ratio of lyoprotectants to yeast biomass is between 0.5:1 and 4:1, preferably between 0.5:1 and 3:1.

Description

FREEZE-DRIED COMPOSITION COMPRISING NON-SACCHAROMYCES YEAST

FIELD

The present application generally relates to the field of yeast, in particular freeze-dried yeast compositions and uses thereof to prepare fermented food and beverage products. Furthermore, the present application relates to processes of manufacturing freeze-dried yeasts.

BACKGROUND

Microorganisms such as yeasts are involved in numerous industrially relevant processes. For instance, yeast cultures are essential in the making of fermented food, including fermented beverages, wine, beer, fermented juice etc. In most cases, it is important that the microorganisms remain viable after prolonged storage in order for these to impart their beneficial effect.

There is a recent and growing interest in the application of non-Saccharomyces yeasts in fermented foods. From the 1990s, interest in non-Saccharomyces yeasts began to increase, and after three decades, the non-Saccharomyces went from being an undesirable yeast associated with spoilage to microorganisms that could improve aromatic profiles of products.

In wine fermentation, co-cultures of non-Saccharomyces yeast of interest with Saccharomyces yeasts are often used for various purposes, such as to generate other aromatic compounds through enzymatic reactions, decrease ethanol content, increase glycerol production, decrease acidity, and stabilize color, etc. In beer, another widely consumed alcoholic beverage, also there is also a growing interest in non-Saccharomyces yeasts, primarily focused on aromatic complexity. This interest is directly related to the development of an artisanal beer market, which seeks to develop products with new and original aromatic notes. In other beverages obtained from different fruits (pineapple, lychee, papaya, bilberry, among others) the possibility of using non-Saccharomyces yeasts has also been studied.

In other fermented foods, such as dairy or dairy analogue products as well as food or beverage prepared from cocoa beans or coffee beans, the use of starter cultures with non-Saccharomyces yeasts have been gaining interests.

WO2011/134952 discloses frozen starter culture comprising non-Saccharomyces wine yeasts for direct inoculation. However, the use of frozen culture requires an unbroken cold chain during transport, which may be expensive and cumbersome. Lyophilization, also known as freeze-drying, is a dehydration method used to preserve cells in a dry state to permit their storage preferably at room temperature. Freeze-drying is a harsh process which negatively affects both viability and physiological state of the yeasts. The formation of ice crystals induces mechanical damage that leads to cellular death during freezing and has impact on viability. For example, survival rates of Saccharomyces yeast have been investigated after freeze-drying and preserving in a vacuum at 5°C. The survival rates of the yeast were only at 10% or less. Miyamoto-Shinohara, Yukie, et al. "Survival rate of microbes after freeze-drying and long-term storage." Cryobiology 41.3 (2000): 251-255.

To our knowledge, non- Saccharomyces yeast has not been satisfactorily preserved yet using freeze- drying techniques at an industrial level. Most documented lyophilization processes involving non- Saccharomyces yeasts have not been scaled to an industrial level.

Thus, there is a need for freeze-dried yeast compositions, in particular non- Saccharomyces yeast compositions since they are becoming increasingly popular as fermenting microorganisms. There is a particular need to provide manufacturing processes which may be scaled to an industrial level and allows good preservation and process survival of yeast cells. Preferably, such compositions exhibit good stability and can be transported without requiring cold chain.

SUMMARY OF THE INVENTION

Lyophilization is a process which is known to trigger thermal, osmotic and oxidative stress in yeasts. The present inventors have discovered that yeast cells can be preserved effectively and with good process storage stability and survivability by using lyoprotectants comprising maltodextrin, antioxidant(s) and disaccharide(s). The weight ratio of the lyoprotectants to yeast (dry matter) in the compositions is in a range preferably between 0.5 and 4. It has been observed that the prepared yeasts have high survivability overthe freeze-drying step, and the obtained compositions are storage stable for prolonged time and can be transported at low temperature or even ambient temperature.

The present application provides freeze-dried yeast compositions, particularly freeze-dried non- Saccharomyces yeast, such as Pichia spp.

A first aspect of the application provides a freeze-dried composition of non-saccharomyces yeast, preferably Pichia spp., comprising a mixture of maltodextrin, antioxidant(s) and disaccharide(s) as lyoprotectants with predetermined weight ratio of the lyoprotectants to yeast biomass (dry matter). This weight ratio is also referred to as encapsulation index (El) in the present application. In preferred embodiments, the weight ratio of the lyoprotectants to yeast biomass is between 0.5:1 and 4:1 , preferably between 0.5:1 and 3:1 in the freeze-dried composition.

In some embodiments, freeze-dried compositions of non-Saccharomyces yeast according to the application comprise from 10⁷ to 10¹² cfu/g of yeast cells, preferably 10⁸ to 10¹¹ cfu/g of yeast cells, more preferably 10⁹ to 10¹° cfu/g of cells.

In some embodiments, freeze-dried compositions of non-Saccharomyces yeast according to the application comprises non-reducing disaccharide which may be trehalose and/or sucrose.

In some embodiments, freeze-dried compositions of non-Saccharomyces yeast according to the application comprises non-reducing disaccharide which may be trehalose, cellulose and/or sucrose. In some embodiments, freeze-dried compositions of non-Saccharomyces yeast according to the application comprises cellulose.

In some preferred embodiments, the lyoprotectants further comprises modified starch.

In some preferred embodiments, the yeast is Pichia kluyveri. In more preferred embodiments, the Pichia kluyveri is DSM 28484 or PK-KR1 .

A second aspect of the application provides uses of the freeze-dried composition of non- Saccharomyces yeast to prepare fermented food products, including fermented dairy or non-dairy products as well as beverages such as wine, beer, juice, cocoa, coffee, etc.

A third aspect of the application provides processes for preparing a freeze-dried non-Saccharomyces yeast composition, comprising providing a yeast cell concentrate, adding lyoprotectants to the yeast cell concentrate to form a suspension, freeze-drying the suspension, characterized in that the lyoprotectants comprise maltodextrin, antioxidant(s) and disaccharide(s) and the dry matter weight ratio of lyoprotectants to yeast biomass is between 0.5:1 and 4:1 , preferably between 0.5:1 and 3:1 , more preferably between 0.7:1 and 2.5:1 .

In preferred embodiments, the application provides a process which comprises:

(1) providing non-Saccharomyces yeast cells,

(2) growing the yeast cells and harvesting the yeast cells to obtain a yeast cell concentrate, wherein the concentrate comprises 10⁸ to 10¹¹ cfu/g dry matter of yeast cells;

(3) adding lyoprotectants to the yeast cell concentrate to form a suspension, wherein the lyoprotectants comprise maltodextrin, antioxidant(s) and disaccharide(s),

(4) freezing the suspension,

(5) drying the suspension, and

(6) obtaining freeze-dried non-Saccharomyces yeast composition. wherein the dry matter weight ratio of lyoprotectants to yeast biomass is between 0.5:1 and 4:1 , more between 0.5:1 and 3:1.

Preferably, the drying is carried out under a vacuum pressure of from 0.01 to 2 millibar(mbar). Preferably, the temperature during drying is controlled such that the vacuum pressure is kept constant.

In some embodiments, the freeze-dried compositions of non-Saccharomyces yeast comprise from 10⁷ to 10¹² cfu/g of yeast cells, preferably 10⁸ to 10¹¹ cfu/g of yeast cells, more preferably 10⁹ to 10¹° cfu/g of yeast cells.

In preferred embodiments, the process further comprises washing the cells after harvesting. In some embodiments, the freeze-dried compositions of non-Saccharomyces yeast comprise nonreducing disaccharide is trehalose or sucrose.

In some embodiments, the freeze-dried compositions of non-Saccharomyces yeast comprise nonreducing disaccharide is trehalose, cellulose, or sucrose.

In some embodiments, the freeze-dried compositions of non-Saccharomyces yeast comprises cellulose.

In some embodiments, the freeze-dried compositions of non-Saccharomyces yeast comprise antioxidants which may be ascorbic acid, citric acid, beta-carotene, vitamin E and glutathione, and/or derivatives thereof, preferably trisodium citrate and sodium ascorbate.

In some preferred embodiments, the composition further comprises modified starch.

In other embodiments, the process further comprises adding excipient(s) to the obtained freeze-dried yeast composition.

A further aspect provides yeast compositions obtained or obtainable by the processes according to the application and uses thereof.

Freeze-dried (FD) yeast composition is particularly advantageous inter alia because offers the benefit that such products can be shipped and stored at anything from -18°C to ambient temperature. The freeze-dried yeast may also be storage stable at -18°C for long periods of time and allow flexible dosing to fit different scale producers.

The present application provides freeze-dried yeast composition which has improved process survival or storage stability than other free-dried compositions known to date.

Furthermore, the inventors also show that the freeze-dried yeasts prepared by the present processes advantageously have similar or improved performances compared to frozen yeast compositions, as demonstrated in Example 6.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows 12 months stability data for Composition 1-7 at storage temperature -18°C.

Figure 2 shows 12 months stability data for Composition 1 -7 at storage temperature 4°C.

Figure 3 shows 12 months stability data for Composition 8 and 10 at storage temperature -18°C.

Figure 4 shows 12 months stability data for Composition 8 and 10 at storage temperature 4°C. Figure 5 shows 6 months stability data for Composition 1 , 6 and 11 at storage temperature -18°C.

Figure 6 shows 6 months stability data for Composition 1 , 6 and 11 at storage temperature 4°C.

Figure 7 shows 6 months stability data for Composition 11 at storage temperature 25°C.

Figure 8 shows 12 months stability data for Composition 12 at storage temperature -18°C.

Figure 9 shows 12 months stability data for Composition 12 at storage temperature 4°C.

Figure 10 shows 12 months stability data for Composition 13-17 storage at temperature 4°C.

Figure 1 1 shows viable cell count of Pichia kluyveri analyzed 1 hour after inoculation in wine.

Figure 12 shows the 3-mercaptohexyl acetate (3-MHA) production of the fermented wine.

Figure 13 shows 12 months stability data for Composition 1 , 6, and 1 1 at storage temperature -18°C.

Figure 14 shows 12 months stability data for Composition 1 , 6, and 11 at storage temperature +4°C.

Figure 15 shows 12 months stability data for Composition 11 at storage temperature +25°C.

DETAILED DESCRIPTION

In general, the terms and phrases used herein have their art-recognized meaning, which can be found by reference to standard texts, journal references, and context known to those skilled in the art. The following definitions are provided to clarify their specific use in context of the disclosure.

As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. As used herein, the term "and/or" means "and" or "or" or both. As used herein, "(s)" after a noun means the plural and/or the singular of the noun.

The terms "comprising", "having", "including" and "containing" are to be construed as open-ended terms (i.e., meaning "including, but not limited to") unless otherwise noted.

The present inventors have worked intensively with numerous different protectants for freeze drying non-Saccharomyces yeasts and identified strategies to freeze-dry the yeast cells which maintain high viability after the process and the product exhibits good storage stability. It has been found that by using a mixture of maltodextrin, antioxidant(s) and disaccharide(s) at certain amounts relative to the yeast cells, the yeast cells can be freeze-dried in a protected manner with high process survival. It has found good results may be achieved when the dry matter weight ratio of lyoprotectants to yeast biomass is preferably between 0.5:1 and 4:1 , more preferably between range of 0.5:1 and 3:1 , most preferably between 0.7:1 to 2.5:1. In a first aspect, the present application provides a freeze-dried composition of non- Saccharomyces yeast, preferably Pichia spp. (such as Pichia kluyveri), comprising maltodextrin, antioxidant(s) and disaccharide(s) (preferably non-reducing) as lyoprotectants, and wherein the dry matter weight ratio of lyoprotectants to yeast biomass is preferably between 0.5:1 and 4:1 .

In preferred embodiments, the application provides freeze-dried compositions wherein the yeast cells are present at a concentration of from 10⁷ to 10¹² cfu/g of yeast cells, preferably 10⁸ to 10¹¹ cfu/g of yeast cells, more preferably 10⁹ to 10¹° cfu/g of cells in the composition.

In preferred embodiments, the freeze-dried composition of non-Saccharomyces yeast, preferably of Pichia spp. (such as Pichia kluyveri), comprises maltodextrin, antioxidant(s) and non-reducing disaccharide(s) as lyoprotectants, and wherein dry matter weight ratio of lyoprotectants to yeast biomass is between 0.7:1 and 2.5:1.

Experimental results demonstrate that freeze-dried yeast compositions have very good results compared to frozen yeast - despite lower amount cells being present initially, aroma production is still comparable or even improved.

Since the composition of the present application is a freeze-dried composition, a skilled person understands that the amounts given for the individual components (e.g. yeast cells and protective agents) of the composition are measured as dry matter. In the present context - the weight of lyoprotectants are given relative to 1 g of yeast cells in the composition. A skilled person in the art is able to prepare the composition using routine methods such that the desired weight ratio is met.

This application relates to freeze-dried yeast, in particular non-Saccharomyces yeast, and especially non-Saccharomyces yeasts that can be used as fermentation microorganisms.

In an embodiment the non-Saccharomyces yeast is selected from Pichia kluyveri, Candida fructus, Citeromyces matritensis, Debaromyces hansenii, Hanseniaspora guillermondii, Hanseniaspora osmophila, Hanseniaspora uvarum, Kluyveromyces marxianus, Kluyveromyces thermotolerans, Pichia fermentans, Pichia membranifaciens, Schizosaccharomyces pombe, Torulaspora delbreuckii, Torulaspora delbrueckii, Zygosaccharomyces bisporus, or Zygosaccharomyces rouxii. Preferably, the non-Saccharomyces yeast is selected from Pichia kluyveri, Pichia membranifaciens, Lachancea thermotolerans, and Torulaspora delbrueckii.

In preferred embodiment, the yeast is Pichia spp. The present application provides in particular a suitable method for formulating Pichia, as this genus is known have an unfavorable response to freeze drying process with low survival rates. Preferred Pichia spp. are Pichia fermentans, Pichia membranifaciens, and Pichia kluyveri. In more preferred embodiments, the yeast is Pichia kluyveri.

In one embodiment, the Pichia kluyveri is the strain deposited as DSM 28484. In another embodiment, the Pichia kluyveri is the PK-KR1 strain disclosed in patent publication W02009110807A1 , deposited as V06/022711 . Microorganisms are often preserved with addition of protective agents (or referred herein as “protectants”) that in various ways may help to stabilize the microorganisms. The term “protective agent(s)” or “protectants” shall herein be understood as any agents that could help to improve the survivability over freeze-drying as well as storage stability of yeast cells of interest. In accordance with the present application, protectants that act to protect during lyophilization (freeze-drying) are termed “lyoprotectants”. The viability protection from the freeze-drying stresses is termed “lyoprotection.”

The present application identifies combined use of antioxidant(s), disaccharide(s) and maltodextrin as suitable lyoprotectants.

One of lyoprotectants used in the present application is antioxidant(s). The term “antioxidant” refers to a compound that inhibits oxidation. The antioxidant may be an industrial chemical or a natural compound. As used herein, antioxidants include, but are not limited to, ascorbic acid, citric acid, beta-carotene, vitamin E and glutathione, and derivatives thereof, especially salts thereof such as trisodium citrate and sodium ascorbate. The term ‘vitamin E’ is to be understood as including any and all variants of tocopherols and tocotrienols (alpha, beta, gamma, delta), whether used alone or together. In a preferred embodiment, the antioxidant is ascorbic acid or salts thereof.

Another one of lyoprotectants used in the present application is disaccharide(s). Disaccharide is formed from two monosaccharides and can be classified as either reducing or non-reducing. According to the present application, the disaccharide is preferably non-reducing disaccharides. Preferably the non-reducing disaccharide is trehalose, cellulose, or sucrose, more preferably trehalose or sucrose, even more preferably trehalose. Preferably the non-reducing disaccharide is trehalose or sucrose.

Another one of lyoprotectants used in the present application is cellulose.

Furthermore, lyoprotectants used here also include maltodextrin. Maltodextrin is a polysaccharide that consists of D-glucose units connected in chains of variable length, typically composed of a mixture of chains that vary from three to 17 glucose units long. The glucose units are primarily linked with a(1 — >4) glycosidic bonds. Maltodextrins typically has a DE (dextrose equivalent) between 5 and 20. As used herein, maltodextrin may include those that have a DE between 5 and 30, preferably between 5 and 25, more preferably between 10 and 20. The higher the DE value, the shorter the glucose chains, the higherthe sweetness, the higherthe solubility, and the lowerthe heat resistance.

It has been found that when the dry matter weight ratio of lyoprotectants to yeast biomass is between 0.5:1 and 4:1 , high viability can be observed. If the weight ratio to too low, adequate protection cannot be conferred. On the other hand, there is a is a diminishing return in protective effects when the weight ratio is too high, with the disadvantages of increased production costs and dilution of cells.

In preferred embodiment, the present application provides freeze-dried compositions of Pichia spp. (such as Pichia kluyveri) comprising maltodextrin, ascorbic acid or salts thereof and trehalose as lyoprotectants, and wherein dry matter weight ratio of lyoprotectants to yeast biomass is between 0.5:1 and 3:1.

In preferred embodiment, the present application provides freeze-dried compositions of Pichia spp. (such as Pichia kluyveri) comprising maltodextrin having a DE between 10 and 20, sodium ascorbate and trehalose as lyoprotectants, and wherein dry matter weight ratio of lyoprotectants to yeast biomass is between 0.7:1 and 2.5:1.

In one preferred embodiment, the compositions further comprise modified starch as lyoprotectants. The term “modified starch” refers to starch that has been physically or chemically altered to improve its functional characteristics. Suitable modified starches include, but are not limited to, pregelatinized starches, low viscosity starches (e.g. dextrins, acid-modified starches, oxidized starches, enzyme modified starches), stabilized starches (e.g., starch esters, starch ethers), cross-linked starches, starch sugars (e.g. glucose syrup, dextrose, isoglucose) and starches that have received a combination of treatments (e.g., cross-linking and gelatinization) and mixtures thereof. In particular, modified starches made from starches substituted by non-chemical methods with hydrophobic moieties are preferred.

The freeze-dried compositions preferably comprise a concentration of viable yeasts in the range from 10⁷ -10¹² CFU/g, preferably at least 10⁸ cfu/g, such as at least 10⁹ cfu/g, such as at least 10¹° cfu/g, and still more preferably at least 5.0 x 10¹° cfu/g of composition. As used herein, the term “viable” in relation to yeast cells refers the ability to form a colony (CFU or Colony Forming Unit) on a nutrient medium appropriate for the growth of the cell. Viable cells are alive and capable of regeneration and/or propagation.

Cell concentration is often assessed by counting the colony-forming units (CFU) per gram, using standard methods known in the art, for example, as set forth in Compendium of international methods of Analysis - OIV, particularly in paragraph 6, CASTELLUCCI, Federico. "Microbiological Analysis of Wines and Musts Detection, Differentiation and Counting of Micro-organisms." (2010).

For example, the formulated and freeze-dried yeast can be suspended in an isotonic solution that does not cause growth of the analyzed cells (i.e. MRD, isotonic salt solution with or without peptone). Mixing may be realized with a stomacher or vortex mixer. This is repeated for multiple serial dilutions, and revitalized in that solution for 30 minutes. The various diluted suspension is then plated on a nutritive medium agar plate (i.e. YM, YEPD, WL Nutrient, TRY, or YGC agar) with a sterile Drigalsky- type rod within 1 to 2 minutes before the liquid is absorbed into the agar surface. The dilutions are chosen that may result in colony counts in the range 10 to 300 per plate. The plates are incubated for 48 to 72 hours at 25-28 °C under aerobic conditions. During that period, the yeast form colonies on the nutritive media. Those colonies were counted and recalculated with the respective dilutions; the results are expressed as Colony Forming Units (CFU) per gram.

The freeze-dried composition can be used as direct vat set (DVS) culture intended for direct inoculation into a fermentation vessel or vat, for the production of fermented food products, including fermented beverages.

The weight of the final product as disclosed herein may be from 1 g to 50kg. Accordingly, it is preferred that the weight of the freeze-dried composition as described herein is from 5g to 30kg, such as from 10g to 40kg or from 50g to 30kg or from 100g to 25kg.

In one preferred embodiment, the composition further comprising excipients added to the freeze- dried yeast. Such excipients may be sucrose, maltodextrin and/or dextrose. As used herein, "excipient" refers to an ingredient that is added to the freeze-dried composition to act as filler.

As illustrated in working examples herein, the freeze-dried compositions as described herein exhibit high process survival and storage stable.

A "stable" composition is one in which the biologically active material therein essentially retains its physical stability, chemical stability, and/or biological activity upon storage. Stability can be measured at a selected temperature and humidity conditions for a predetermined time of period. Trend analysis can be used to estimate an expected shelf life before a material has actually been in storage for that time period. For yeast cells, for example, storage stability may be defined as the time it takes to lose 1 log of CFU/g dry formulation under predetermined conditions of temperature, humidity and time period.

Storage stability can be determined by analyzing how the count of viable microbial cells develops overtime. Viability of the microbial culture is measured by determining the CFU/g as described herein. Thus, a measure of the storage stability of freeze-dried yeast may be determined by placing a sample with a water activity (aw) of 0.15 or less in a sealed laminated aluminum foil and evaluating CFU/g of the yeasts at time point 0 (just after drying) and after 6 months storage at 4°C.

Process survival, also herein referred to as survivability, indicates the extent of cell survival after freeze-drying. This can be represented as log loss: log differences between CFUs of 100 % recovery (theoretical) and the actual measured CFU (measured). Survival can also be represented as decrease between CFUs of 100 % recovery and the actual measured CFU in percentage. At least 30% process survival can be achieved with the presently disclosed methods. Use of composition

In a further aspect, the present application also provides use of the freeze-dried prepare fermented food products. Any fermented food products for which the yeasts can be used are included herein. In present application, the preparation of beverages such as wine, beer, juice (vegetable juice or fruit juice), cider, kefir, cocoa, coffee and the like is particularly relevant. Yeast compositions of the present application may be rehydrated before inoculation. In another preferred embodiment, the yeast is not rehydrated before inoculation.

Process

One main aspect of the present application provides a process for preparing a freeze-dried non- Saccharomyces yeast composition, wherein the process comprises the following steps:

(1) providing non-Saccharomyces yeast cells, preferably Pichia spp.,

(2) growing the yeast cells and harvesting the yeast cells to obtain a yeast cell concentrate, wherein the concentrate comprises 10⁸ to 10¹¹ cfu/g dry matter of yeast cells,

(4) freezing the suspension,

(5) drying the suspension, and

(6) obtaining freeze-dried non-Saccharomyces yeast composition. wherein the dry matter weight ratio of lyoprotectants to yeast biomass is between 0.5:1 and 4:1 , preferably between 0.5:1 and 3:1.

In a preferred embodiment, the application provides a process for preparing a freeze-dried non- Saccharomyces yeast composition, wherein the process comprises the following steps:

(1) providing non-Saccharomyces yeast cells,

(3) adding lyoprotectants to the yeast cells concentrate to form a suspension, wherein the lyoprotectants comprise maltodextrin, antioxidant(s) and (preferably non-reducing) disaccharide(s),

(4) freezing the suspension,

(5) drying the suspension, and

(6) obtain freeze-dried non-Saccharomyces yeast composition. wherein the dry matter weight ratio of lyoprotectants to yeast biomass is between 0.5:1 and 3:1 , preferably between 0.7:1 to 2.5:1 and wherein the antioxidant is ascorbic acid or salts thereof and the disaccharide is trehalose.

To prepare the freeze-dried composition of the application, a non-Saccharomyces yeast is provided to prepare a concentrate of yeast cells. In this, the process involves fermenting the yeast cells and harvesting the yeast cells to obtain a yeast cell concentrate which comprises 10⁸ to 10¹¹ cfu/g dry matter of yeast cells.

It is routine work for the skilled person to grow a non-Saccharomyces yeast cell of interest in order propagate it in large scale. Propagation is typically done in a medium which would depend on the nutrient requirements of particular the non-Saccharomyces yeast intended. As used herein, the term “grow” or “growing” refers to the generation of yeast biomass.

As known in the art, harvesting of cells generally involves a centrifugation step to remove parts of the fermentation media and obtain get a yeast cell concentrate. Alternative methods such filtration are also possible.

The obtained yeast cell concentrate may comprise from 10⁸ to 10¹¹ cfu/g dry matter of the concentrate of yeast cells”. The term “dry matter” with the term “cfu/g dry matter” should be understood as the skilled person would understand it in the present context - i.e. that the yeast concentrate comprises the given amount of yeast cells as relative to the dry matter weight of the yeast concentrate.

In one preferred embodiment, after the harvesting of the cell, it may be preferred to include an extra washing step in order to remove the fermentation media components and to condition the cells. Washing the cells before drying is known in the art and has been described for example in EP Patent No. 0189318. In preferred embodiments, washing can be performed by using a water-based solution containing magnesium and calcium salts.

After a yeast cell concentrate is obtained, lyoprotectants are added typically as a liquid solution to form a suspension. As described in the present application, the lyoprotectants comprise maltodextrin, antioxidant(s) and disaccharide(s).

The disaccharide(s) is preferably non-reducing disaccharides. Preferably the non-reducing disaccharide is trehalose, cellulose, or sucrose, more preferably trehalose or sucrose, even more preferably trehalose. In some embodiments, the disaccharide is reducing disaccharide(s), such as lactose, isomaltose, maltose, palatinose and cellobiose, more preferably maltose. Suitable antioxidants include, but are not limited to, ascorbic acid, citric acid, beta-carotene, vitamin E and glutathione, and derivatives thereof, especially salts thereof such as trisodium citrate and sodium ascorbate.

Suitable maltodextrins may have a DE between 5 and 30, between 5 and 25, preferably between 10 and 20.

As described earlier in this application, the dry matter weight ratio of lyoprotectants to yeast biomass is preferably between 0.5:1 and 4:1 . It is routine work for the skilled person to formulate the yeast cell concentrate correctly to reach the desired weight ratio as described herein.

Other lyoprotectants can also be added to the yeast cell concentrate. For example, modified starch can be included. Thus, in preferred embodiments, the lyoprotectants further comprise modified starch

After lyoprotectants are added, the formulated yeast cells are subject to lyophilization (freeze-drying). Typical lyophilization process includes (1) freezing, wherein water is converted into ice and (2) drying, wherein ice is removed by sublimation and unfrozen water is removed by desorption. Freeze-drying methods are known in the art and for example described in Labconco, I. "A Guide to Freeze Drying for the Laboratory." (2008).

Thus, the presently disclosed freeze-dried composition is prepared by first freezing and then drying the yeast cells in the formulated suspension. Preferably, formulated yeast cells are transferred to a suitable container such as a tray before freezing. It is routine work for the skilled person to carry out the freezing step and in the process selecting suitable parameters, e.g. pressure, temperature and time. Freezing is typically done below -10°C, preferably below -20°C, below -30°C, below -40°C, below -50°C. As known to a skilled person in the art, this temperature should not exceed the glass transition temperature of the solution, as otherwise melting, which impacts the yeast physiology, would occur. Freezing can be carried out in a freezer.

In some embodiments, the suspension comprising the yeast cells is frozen in liquid nitrogen to form pellets. This is also known as pelletizing. The term "pellet" refers to small, solid granules. The term "pelleting" refers to the processes for forming such solid granules. Pelletizing methods are known in the art. One method may be to let drops of the mixture fall into liquid nitrogen. Another method for forming pellets may involve spray cooling the concentrate.

In preferred embodiments, the formulated yeast cells are frozen in a tray. For industrially relevant large-scale production, it is preferred to simultaneously use multiple trays, such as more than 10 or more than 100 trays. After freezing, the yeast cells are subject to drying in the second phase of lyophilization. In this phase, dehydration of the yeasts takes place through the sublimation. A skilled person knows how to select suitable drying parameters in the lyophilization process, which typically involves low temperature and reduced pressure.

In preferred embodiments, drying is carried out at a temperature below -10°C, preferably below - 20°C or below -30°C. Preferably, the temperature during drying is controlled such that a predetermined vacuum pressure is kept constant.

In preferred embodiments, drying is carried out at a vacuum pressure of from 0.01 to 2 millibar(mbar). More preferably, it is carried out is carried out at a vacuum pressure of from 0.5 to 2 mbar, such as 0.7 to 2 mbar.

In preferred embodiments, drying is carried out for a period of time sufficient to reduce the water activity (aw) to less than 0.20 and more preferably to less than 0.15. Drying may be terminated when constant weight is attained. Water activity is a well-known parameter and is the partial vapor pressure of water in a substance divided by the standard state partial vapor pressure of water. Herein, the standard state is defined as the partial vapor pressure of pure water at the same temperature. Using this definition, pure distilled water has a water activity of exactly one. The skilled person knows how to determine the water activity of the composition. For example, suitable methods for measuring aw are set out on the FDA website under the heading “Water Activity (aw) in Foods”, as published on 27 August 2014.

In preferred embodiments, an apparatus that is able to carry out both freezing and drying, such as a freeze-dryer, is used.

A freeze-dried non-Saccharomyces yeast composition can be obtained after the drying step.

The freeze-dried yeasts may be subject to further treatment such mechanically reduced in size, for example milled or ground into smaller particles, such as between 200-1000pm, prior to formulation of the final product.

As understood by the skilled person, afterthe freeze-drying, the method may comprise optional steps to obtain the final product.

As used herein, the term "product" in certain instances may also contain other components, including excipients or other microorganisms. In one preferred embodiment, the process further comprising adding excipients to the freeze-dried yeast composition to obtain a final product. Such excipients may be sucrose.

A herein relevant optional extra step would be to e.g., properly packaging the obtained freeze-dried yeast composition. The package may e.g., be a bottle, box, bag, etc. The package should be able to keep the water activity of the composition low. In the present context, the term “packaging” a suitable amount of the freeze-dried microorganism in a suitable package relates to the final packaging to obtain a product that can be shipped to a customer. A suitable package may thus be a container, bottle or similar, and a suitable amount may be e.g. 10g to 50kg. Such yeast composition is highly useful. The composition may be directly inoculated into the fermentation medium without intermediate propagation steps.

A further aspect of the present application is the composition obtained by the process of the application as described herein. In one preferred embodiment, obtained composition comprises 10⁷ to 10¹¹ cfu/g yeast cells. In another embodiment, obtained composition comprises 10⁸ to 10¹¹ cfu/g yeast cells. In yet another embodiment, obtained composition comprises 10⁹ to 10¹¹ cfu/g yeast cells.

DEPOSIT AND EXPERT SOLUTION

The applicant requests that a sample of the deposited microorganisms stated below may only be made available to an expert, subject to available provisions governed by Industrial Property Offices of States Party to the Budapest Treaty, until the date on which the patent is granted.

The applicant has made the following deposits at a Depositary institution having acquired the status of international depositary authority under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure: Leibniz Institute DSMZ- German Collection of Microorganisms and Cell Cultures Inhoffenstr. 7B, 38124 Braunschweig, Germany.

Strain Accession No. Deposit date

Pichia kluyveri DSM 28484 DSM 28484 2014.3.5

EXAMPLES

The present application has been described with reference to various embodiments, aspects, examples, or the like. Itis not intended that these elements be read in isolation from one another. Thus, the present disclosure provides for the combination of two or more of the embodiments, aspects, examples, or the like.

All embodiments described herein are intended to be within the scope of the application disclosed. These and other embodiments of the present application will become readily apparent to those skilled in the art from the following detailed description of the embodiments having reference to the whole description, the application not being limited to any particular embodiments disclosed.

Several freeze-dried compositions of non-Saccharomyces (Pichia kluyveri) were prepared, using different formulations and amount of lyoprotectants.

Example 1 Freeze-dried Pichia kluyveri composition using PK-KR1 (V06/022711)

This example shows several freeze-dried yeast compositions 1-7. The Pichia kluyveri yeast cell used was PK-KR1 . 1.1 Preparations of the compositions

Yeast cell concentrate

The yeast cells were grown and harvest as follows:

The yeast was grown using a submerged fermentation process in a liquid complex nutrient medium in a closed fermentation vessel. The yeast biomass was harvested by discharge or continuous separation. The harvested and concentrated yeast biomass comprises around 17 % dry matter and 83 % of water.

Table 1 summarizes the total amount of wet biomass to be formulated, dry matter of wet biomass, wash of biomass before formulation, total amount of lyoprotectants used and encapsulation index. Table 1

Formulation of compositions

Briefly, 3 different lyoprotectant stock solutions containing disaccharide, antioxidant, maltodextrin, with or without modified starch were prepared. Solution 1-3 all contain trehalose, sodium ascorbate, and maltodextrin, and solution 2 additionally contains modified starch. Different amount of the solution was added to the Pichia kluyveri biomass to reach the encapsulation index (ratio of lyoprotectant dry matter/yeast dry matter).

More details are provided below:

Composition 1

Composition 1 acts as a control and no lyoprotectants were added to the biomass. The wet biomass had a potency of 7.15E+09 CFU/g and was kept at 5°C before transferred to a small tray in the freeze dryer.

Composition 2

Concentrated biomass was formulated with lyoprotectant solution 1 ensuring that 200 g wet concentrated biomass was supplemented in total by 34.4 g trehalose, 15.1 g sodium ascorbate and 20 g and maltodextrin (Glucidex IT12, DE=12) resulting in an encapsulation index corresponding to 2 (total 69.5 g dry lyoprotectant and 34 g dry biomass). The formulated concentrate was kept at 5°C and mixed for 20 minutes before transferred to a tray in the freeze dryer.

Composition 3

Concentrated biomass was formulated with lyoprotectant solution 1 ensuring that 200 g wet concentrated biomass was supplemented in total by 25.8 g trehalose, 11 .3 g sodium ascorbate and 14.9 g maltodextrin resulting in an encapsulation index corresponding to 1.5. The formulated concentrate was kept at 5°C and mixed for 20 minutes before transferred to a tray in the freeze dryer.

Composition 4

Concentrated biomass was formulated with lyoprotectant solution 2 ensuring that 200 g wet concentrated biomass was supplemented in total by 31 .4 g trehalose, 5.1 g sodium ascorbate, 2.4 g maltodextrin and 30.5 g modified starch HICAP 100 (Ingredion, Westchester, IL) resulting in an encapsulation index corresponding to 2. The formulated concentrate was kept at 5°C and mixed for 20 minutes before transferred to a tray in the freeze dryer.

Composition 5

Concentrated biomass was formulated with lyoprotectant solution 2 ensuring that 200 g wet concentrated biomass was supplemented in total by 23.4 g trehalose, 3.8 g sodium ascorbate, 1 .8 g maltodextrin and 22.8 g modified starch HICAP 100 resulting in an encapsulation index corresponding to 1 .5. The formulated concentrate was kept at 5°C and mixed for 20 minutes before transferred to a tray in the freeze dryer.

Composition 6

Concentrated biomass was formulated with lyoprotectant solution 3 ensuring that 200 g wet concentrated biomass was supplemented in total by 23.3 g trehalose, 3.1 g sodium ascorbate and 42.9 g maltodextrin resulting in an encapsulation index corresponding to 2. The formulated concentrate was kept at 5°C and mixed for 20 minutes before transferred to a tray in the freeze dryer.

Composition 7

Concentrated biomass was formulated with lyoprotectant solution 3 ensuring that 200 g wet concentrated biomass was supplemented in total by 17.5 g trehalose, 2.3 g sodium ascorbate and 32.3 g maltodextrin resulting in an encapsulation index corresponding to 1.5. The formulated concentrate was kept at 5°C and mixed for 20 minutes before transferred to a tray in the freeze dryer.

Freeze-drying

The samples were transferred onto precooled trays in a 10 kg freeze-dryer (HOF Sonderanlagenbau GmbH, Germany) and frozen slowly to -50°C over a period of 3 hrs (<2K/min) and kept at -50°C for additional 2 hrs. The freeze-dryer chamber was subsequently adjusted to between 0.01 - 2.00 mbar over a time period of approx. 45 minutes. The remaining primary drying took place over a period of 30 hrs where the temperature slowly increased from -50°C to 0 °C. Secondary drying was carried for 15 hrs at +25°C. Freeze-dried yeasts were removed from the tray and ground into power.

The final freeze-dried yeast concentration (CFU/g) for each of the final product was measured and shown in Table 1 .

1.2 Evaluation of process survival and storage stability based on CFU/a

Process survival

Survival over freeze drying indicates the extent of cell survival after freeze-drying. This is represented as log loss: log differences between CFUs of 100 % recovery (theoretical) and the actual measured CFU (measured). The expected theoretical CFU value can be calculated knowing the CFU/g of the concentrate and multiplying with the obtained dilution factor when formulated and the concentration factor obtained during the freeze-drying process (FD). Survival can also be represented as decrease between CFUs of 100 % recovery and the actual measured CFU in percentage.

Storage stability

Long term stability of freeze-dried cultures as a function of time is a critical characteristic for commercial application. The long-term stability was evaluated by placing the freeze-dried cells into a sealed laminated aluminum foil in a constant temperature chamber maintained at a predetermined temperature (25°C, 4°C or -18°C) for a predetermined period of time (up to 12 months). The cell count of the freeze-dried material was measured before and after the exposure to the predetermined temperature and time. Difference in CFU is represented as log loss, calculated by subtracting the logarithmic value of the initial measured cells from cells measured at end of the stability test.

1.3 Results

Results of process survival Table 2 shows the calculated process survival over the freeze-drying step.

Table 2

Results of storage stability Table 3 shows the storage stability data of the Composition 1- 7 after 12 months of storage.

Table 3

In addition, Figure 1 and 2 show the stability data of the compositions throughout the period of 12 months at -18°C and 4°C, respectively. 1.4 Conclusion

Process Survival

As shown in the results from Table 2, it is possible to reduce the log losses over the freeze-drying step and thus to increase the process survival using the tested lyoprotectant solutions. In addition, it was found that higher encapsulation index resulted in lower log losses. For instance, using lyoprotectant 2 and increasing the encapsulation index from 1 .5 to 2, (Composition 5 and 4) it was possible to further reduce the log losses from 0.42 to 0.28. Similarly, for Composition 7 and 6, where the encapsulation index increased from 1 .5 to 2 using lyoprotectant solution 3, it was possible to reduce the log losses from 0.3 to 0.13. The poorest survival over the freeze-drying step was observed in control, where no lyoprotectants (El = 0) were used (Composition 1). There, the survival over the freeze-drying step was about 25 %.

Storage Stability

As shown in the results from Table 3 and Figure 1 and 2, stability of the freeze-dried Pichia kluyveri was significantly improved when using the tested lyoprotectants. Poorest stability after 12 months of storage at -18°C and 4°C was observed for the freeze-dried product without lyoprotectants (Composition 1). The log losses were 0.32 for the product stored at -18°C and 1.33 for the product stored at 4°C. No log losses were observed for the freeze-dried products containing lyoprotectants store at -18°C. Furthermore, the log losses for Composition 2-7 stored at 4°C were all significantly lower than to Composition 1 stored at same temperature.

Example 2 Freeze-dried Pichia kluyveri composition using PK-KR1 (V06/022711)

This example shows several freeze-dried non-Saccharomyces yeast Pichia kluyveri Compositions 8-10. The Pichia kluyveri yeast cell was PK-KR1 .

2.1 Preparations of the compositions

The compositions were prepared essentially the same way as described in Example 1 , except that only stock solution 1 was used for preparing Composition 9-10. The harvested and concentrated yeast biomass comprises around 17.4 % dry matter and 82.6 % of water.

Table 4 summarizes the total amount of wet biomass to be formulated, dry matter of wet biomass, wash of biomass before formulation, total amount of lyoprotectants used and encapsulation index.

Table 4

Formulation of the compositions

Composition 8

Composition 8 served as a control and no lyoprotectants were added to the biomass. The concentrated wet biomass had a potency of 1 .09E+10 CFU/g and was kept at 5°C before transferred to a small tray in the freeze dryer.

Composition 9

Concentrated biomass was formulated with lyoprotectant solution 1 ensuring that 200 g wet concentrated biomass was supplemented in total by 12.7 g trehalose, 6 g sodium ascorbate and 7.5 g maltodextrin resulting in an encapsulation index corresponding to 0.75 (total 26.2 g dry lyoprotectant and 34.8 g dry biomass). The formulated concentrate was kept at 5°C and mixed for 20 minutes before transferred to a tray in the freeze dryer.

Composition 10

Concentrated biomass was formulated with lyoprotectant solution 1 ensuring that 200 g wet concentrated biomass was supplemented in total by 19.4 g trehalose, 8.5 g sodium ascorbate and 11.2 g maltodextrin resulting in an encapsulation index corresponding to 1.1. The formulated concentrate was kept at 5°C and mixed for 20 minutes before transferred to a tray in the freeze dryer.

The final freeze-dried yeast concentration (CFU/g) for each of the final product was measured and shown in Table 4. 2.2 Results

Process survival and storage stability was evaluated as in Example 1 .

Results of process survival

Table 5 shows the calculated process survival over the freeze-drying step for Composition 8-10.

Table 5

Results of storage stability

Table 6 shows the storage stability data of the Composition 8 and 10 after 12 months of storage.

This was not measured for Composition 9 and thus the data was not available.

Table 6

In addition, Figure 3 and 4 show the stability data for Compositions 8 and 10 throughout the period of 12 months at -18°C and 4°, respectively.

2.3 Conclusion

Process Survival

As shown in the results from Table 5, it is possible to reduce the log losses over the freeze-drying step and thus increase the process survival using the tested lyoprotectant solutions. In addition, it was found that by increasing the encapsulation index from 0 up to 0.75 and 1.1 , it was possible to reduce the log losses from 0.82 to 0.47 and 0.35, respectively. The poorest survival over the freeze- drying step was observed in control, where no lyoprotectants were used (Composition 8). Survival over the freeze-drying step was about 15 %, compared to 33 % and 44 % in Composition 9 and 10, respectively, where the encapsulation index was 0.75 and 1 .1 , respectively. Storage Stability

As shown in the results from Table 6 and Figure 3 and 4, stability of the freeze-dried Pichia kluyveri was significantly improved when using lyoprotectants. Poorest stability after 12 months of storage at -18°C and 4°C was observed for the freeze-dried product without lyoprotectants (Composition 8). Here, the log losses were 0.47 for the product stored at -18°C and 1 .51 for the product stored at 4°C. No log losses were observed for the freeze-dried products with the highest encapsulation index (Composition 10) store at -18°C. The log losses for Composition 10 stored at 4° was significantly lower than Composition 8 stored at same temperature. Example 3 Freeze-dried Pichia kluyveri composition using PK-KR1 (V06/022711) with storage stability data for -18 °C, 4°C and 25°C

3.1 Preparation of the compositions

Composition 11 was prepared essentially the same way as Composition 6 described in Example 1 . Lyoprotectant solution 3 was used. The harvested and concentrated yeast biomass comprises 15.6 % dry matter and 84.4 % of water.

Table 7 summarizes the total amount of wet biomass to be formulated, dry matter of wet biomass, wash of biomass before formulation, total amount of lyoprotectants used and encapsulation index.

Table 7

Formulation of the composition

Composition 11

Concentrated biomass was formulated with lyoprotectant solution 3 ensuring that 200 g wet concentrated biomass was supplemented in total by 23.3 g trehalose, 3.1 g sodium ascorbate and 42.9 g maltodextrin resulting in an encapsulation index corresponding to 2.2 (total 69.3 g dry lyoprotectant and 31.2 g dry biomass). The formulated concentrate was kept at 5°C and mixed for 20 minutes before transferred to a tray in the freeze dryer.

The final freeze-dried yeast concentration (CFU/g) was measured and is shown in Table 7.

3.2 Results

Process survival and storage stability was evaluated as in Example 1 .

Results of process survival

Table 8 shows the calculated process survival over the freeze-drying step for Composition 11 .

Table 8

Results of storage stability

Table 9 shows the storage stability data of Composition 11 after 6 months of storage at -18°C, 4°C and 25°C.

Table 9

In addition, Figure 5, 6 and 7 show the stability data for Composition 11 throughout the period of 6 months stored at -18°C, 4°C and 25°C, respectively. Composition 1 (control, no use of lyoprotectants) and Composition 6 (same lyoprotectants and approximately the same El as Composition 11) are included in the Figure 5 and 6 for comparison.

Table 10

In addition, Figure 13, 14 and 15 show the stability data for Composition 11 throughout the period of 12 months stored at -18°C, 4°C and 25°C, respectively. Composition 1 (control, no use of lyoprotectants) and Composition 6 (same lyoprotectants and approximately the same El as Composition 11) are included in the Figure 13 and 14 for comparison.

3.3 Conclusion

Process Survival

As shown in the results from Table 8, it is possible reduced the log losses over the freeze-drying step and thus increase the process survival, comparing to Composition 1 (no lyoprotectants used, described in example 1). The process survival achieved over the freeze-drying step for Composition 11 is furthermore reproducible and comparable with similar Composition 6 as described in Example 1. The process survival was approx. 0.13 and 0.14 for Composition 6 and Composition 11 , respectively.

Storage Stability

As shown in the results from Table 9 and Figure 5 through 7, stability of the freeze-dried Pichia kluyveri was significantly improved when using lyoprotectants. For comparison, stability data for composition 1 and 6 were included in Figure 5 and 6. It can be seen that the stability profile for both are Composition 6 and 11 are very similar.

Even at 25°C, stability data shown in Figure 7 demonstrated improved stability for Composition 11 than Composition 1 stored and measured at 4°C. Example 4 Freeze-dried Pichia kluyveri composition using PK-KR1 (V06/022711)

4.1 Preparation of the composition

Composition 12 was prepared essentially the same way as Composition 10 described in Example 2, using lyoprotectant solution 1 , except that the non-Saccharomyces yeast Pichia kluyveri biomass before drying had been washed in solution containing magnesium and calcium salts. The washed yeast biomass comprises around 21 .1 % dry matter and 78.9 % of water.

Table 16 summarizes the total amount of wet biomass to be formulated, dry matter of wet biomass, wash of biomass before formulation, total amount of lyoprotectants used and encapsulation index. The biomass was washed in a solution of calcium chloride and magnesium chloride as a pretreatment (79 g/L CaCI 2xH2O, 109 g/L MgCI 6XH2O). Washing was carried out using 400 g of concentrated biomass in 1 .4:1 (biomass to salt solution ratio) mixture with above mentioned water-based solution containing magnesium and calcium salts for 30 min. Afterwards it was concentrated on a centrifuge removing an amount of supernatant equal to the salt solution added before. The lyoprotectants were hereafter added to the washed biomass before freeze-drying. Table 16

Formulation of the composition

Composition 12

Concentrated washed biomass was formulated with lyoprotectant solution 1 ensuring that 200 g wet concentrated biomass was supplemented in total by 23 g trehalose, 10 g sodium ascorbate and 13.3 g maltodextrin resulting in an encapsulation index corresponding to 1 .1 . The formulated concentrate was kept at 5°C and mixed for 20 minutes before transferred to a tray in the freeze dryer.

The final freeze-dried yeast concentration (CFU/g) was measured and is shown in Table 16.

4.2 Results

Process survival and storage stability was evaluated as in Example 1 .

Results of process survival

Table 16 shows the calculated process survival over the freeze-drying step for Composition 12.

Table 16

Results of storage stability

Table 16 shows the storage stability data of Composition 12 after 12 months of storage at -18°C and

4°C.

Table 16

In addition, Figure 8 and 9 show the stability data for Composition 12 throughout the period of 12 months stored at -18°C and 4°C, respectively. 4.3 Conclusion

Process Survival

The results from Table 16 demonstrate that washing of the biomass has a high impact on the survival of the yeast over the freeze-drying step. The log loss was calculated to 0.004, corresponding to approximately a survival on 98 %. The survival is significantly higher than the survival obtained with non-washed biomass as tested in Composition 10 in Example 2. There, the survival was calculated to 44 %.

Storage Stability

As shown in the results from Table 16, Figure 8 and 9, it is also evident that stability of the freeze- dried Pichia kluyveri can be maintained after washing the biomass before drying and formulated using lyoprotectant solution 1 . No log loss was obtained for the freeze-dried Pichia kluyveri product stored at -18°C after 12 months of storage. Log loss after 12 months of storage at 4°C was measured to 0.12 - slightly better compared to results obtained for the non-washed biomass (Composition 10) described in Example 2.

Example 5 Freeze-dried Pichia kluyveri composition comprising DSM 28484

This example shows several freeze-dried non-Saccharomyces yeast Compositions 13-17, using another strain of Pichia kluyveri.

5.1 Preparation of the compositions

The compositions were prepared in the same way as described in Example 1 , except the yeast cell formulated was DSM 28484. The harvested and concentrated yeast biomass comprises around 17.5 % dry matter and 82.5 % of water.

Table 16 summarizes the total amount of wet biomass to be formulated, dry matter of wet biomass, wash of biomass before formulation, total amount of lyoprotectants used and encapsulation index.

Table 16

Formulation of the compositions

Composition 13

Composition 13 acts as control and no lyoprotectants were added to the biomass. The wet biomass had a potency of 1 .37E+10 CFU/g and was kept at 5°C before transferred to a small tray in the freeze dryer.

Composition 14

Concentrated biomass was formulated with lyoprotectant solution 4 ensuring that 200 g wet concentrated biomass was supplemented in total by 84 g trehalose resulting in an encapsulation index corresponding to 2.4 (total 84 g dry lyoprotectant and 35 g dry biomass). The formulated concentrate was kept at 5°C and mixed for 20 minutes before transferred to a tray in the freeze dryer.

Composition 15

Concentrated biomass was formulated with lyoprotectant solution 2 ensuring that 200 g wet concentrated biomass was supplemented in total by 32.7 g trehalose, 5.3 g sodium ascorbate, 2.5 g maltodextrin and 31.8 g modified starch HICAP 100 resulting in an encapsulation index corresponding to 2.1 . The formulated concentrate was kept at 5°C and mixed for 20 minutes before transferred to a tray in the freeze dryer.

Composition 16

Concentrated biomass was formulated with lyoprotectant solution 1 ensuring that 200 g wet concentrated biomass was supplemented in total by 34.7 g trehalose, 15.6 g sodium ascorbate and

20.7 g maltodextrin resulting in an encapsulation index corresponding to 2. The formulated concentrate was kept at 5°C and mixed for 20 minutes before transferred to a tray in the freeze dryer.

Composition 17

Concentrated biomass was formulated with lyoprotectant solution 3 ensuring that 200 g wet concentrated biomass was supplemented in total by 24.3 g trehalose, 3.2 g sodium ascorbate and

44.7 g maltodextrin resulting in an encapsulation index corresponding to 2.1. The formulated concentrate was kept at 5°C and mixed for 20 minutes before transferred to a tray in the freeze dryer. The final freeze-dried yeast concentration (CFU/g) for each of the final product was measured and shown in Table 16.

5.2 Results

Process survival and storage stability were measured as in Example 1 .

Results of process survival

Table 16 shows the calculated process survival over the freeze-drying step for Composition 13-17.

Table 16

Results of storage stability

Table 16 shows the storage stability data of Composition 13-17 after 12 months of storage at 4°C.

Table 16

In addition, Figure 10 shows the stability data of Composition 13-17 throughout the period of 12 months stored at 4°C.

5.3 Conclusion

Process Survival

As shown in the results from Table 16, it confirms the finding in Example 1 that it is possible to reduce the log losses over the freeze-drying step and thus increase the process survival using the tested lyoprotectant solutions. In addition, it was found that higher encapsulation index resulted in lower log losses. For instance, by using lyoprotectant solution 2 and an encapsulation index of 2.1 (Composition 15), it is possible to reduce the log losses from 0.71 (control Composition 13) to 0.22. Using trehalose alone (Composition 14) also improved process survival. The poorest survival over the freeze-drying step was observed for the Pichia kluyveri where no lyoprotectants are used (Composition 13). Survival over the freeze-drying step was estimated to 20 %. Storage Stability

As shown in the results from Table 16 and Figure 10, it also follows that stability of the freeze-dried Pichia kluyveri can significantly improve when using all lyo protectants according to the application. However, stability of the freeze-dried Pichia kluyveri is lost by using trehalose alone (Composition 14). Log losses was more than 1 unit after 12 months of storage at 4°C. It has been observed that using trehalose alone resulted in a freeze-dried final product with higher degree of meringue formation compared to Composition 15 - 17, suggesting that higher degree of melting had taken place during the freeze-drying process. Best stability was obtained by replacing part of the trehalose with antioxidant and high amount of modified starch (Composition 15) or by adding antioxidant and high amount of maltodextrin (Composition 16 and 17).

Example 6 Use of freeze-dried non-Saccharomyces to ferment wine

6.1 Fermentation of wine

Frozen or freeze-dried Pichia kluyveri prepared in Example 3 was used to prepare German Chardonnay must in sequential inoculation. In detail, Pichia kluyveri (freeze-dried or frozen) was inoculated first, and two days later a second yeast Saccharomyces cerevisiae was inoculated. The production of 3-mercaptohexyl acetate (3-MHA), a volatile thiol which imparts a passion fruit and tropical aroma, that is known to be produced by the yeast was measured.

Each fermentation was performed on 5 liters pasteurized German Chardonnay must from grapes harvested in 2022. Both the freeze dried and the frozen Pichia kluyveri were inoculated directly without any pre-treatment. The same Saccharomyces cerevisiae yeast were inoculated directly to all fermentations in the same concentration after 2 days. All fermentations were stabilized with 40 ppm SO2 and bottled after alcoholic fermentation was finished (total glucose/Fructose < 1 g/L).

Viable cell count: total yeast count was analyzed 1 hour after inoculation by spread-plating samples on YGC media and incubating at 20°C for 72 hours.

Thiol concentration: 3-mercaptohexyl acetate (3-MHA) concentration in the final wine was measured by determination of polyfunctional mercaptans based on a simultaneous derivatization and extraction method with DCM followed by LC-QqQ analysis, based on what was published in S. Vichi, N. Cortes- Francisco, J. Caixach. Analysis of volatile thiols in alcoholic beverages by simultaneous derivatization/extraction and liquid chromatography-high resolution mass spectrometry. Food Chemistry. Vol. 175. May 2015, Pages 401-408.

6.2 Results

Wine fermented with freeze-dried Pichia kluyveri had a higher concentration of 3-MHA than the wine fermented with frozen Pichia kluyveri, despite the cell number detected initially being lower. The freeze-dried Pichia kluyveri surprisingly led to higher amount of 3-MHA production. This suggests that the freeze-dried format was able to result in wine of comparable quality that from the frozen yeast.

Claims

1 . A freeze-dried composition of non-Saccharomyces yeast, preferably Pichia spp. or Pichia kluyveri, comprising maltodextrin, antioxidant(s) and disaccharide(s) as lyoprotectants, and wherein dry matter weight ratio of lyoprotectants to yeast biomass is between 0.5:1 and 4:1 .

2. The freeze-dried composition according to claim 1 , wherein the disaccharide is non-reducing disaccharide, preferably trehalose or sucrose, and/or wherein the antioxidant is ascorbic acid, citric acid, beta-carotene, vitamin E and glutathione, and/or derivatives thereof.

3. The freeze-dried composition according to any one of claims 1-2, further comprising modified starch.

4. The freeze-dried composition according to any one of claims 1-3, wherein the maltodextrin has a dextrose equivalent (DE) between 5 and 30, preferably between 10 and 20.

5. The freeze-dried composition according to any one of claims 1 -4 comprising from 10⁷ to 10¹² cfu/g yeast cells.

6. The freeze-dried composition according to any of any one of claims 1-5, wherein the yeast is Pichia spp., preferably Pichia kluyveri.

7. The freeze-dried composition according to claim 6, wherein the Pichia kluyveri is DSM 28484 or PK-KR1 .

8. The freeze-dried composition according to any one of claims 1-7, further comprising excipients.

9. Use of the freeze-dried composition according to any one of claims 1-8 to prepare fermented food products, preferably beverages.

10. A process for preparing a freeze-dried composition of non-Saccharomyces yeasts, comprising the following steps:

(1) providing non-Saccharomyces yeast cells,

(4) freezing the suspension,

(5) drying the suspension, and

(6) obtaining freeze-dried non-Saccharomyces yeast composition. wherein the dry matter weight ratio of lyoprotectants to yeast biomass is between 0.5:1 and 4:1 , preferably between 0.5:1 and 3:1 , more preferably between 0.7:1 to 2.5:1 .

11 . The process according to claim 10, wherein the obtained composition comprises 10⁹ to 10¹³ cfu/g yeast cells.

12. The process according to any one of claims 10-11 , wherein the drying is carried out at a vacuum pressure of from 0.01 to 2 millibar(mbar) and/or the temperature during drying is controlled such that the vacuum pressure is kept constant.

13. The process according to any one of claims 10-12, wherein in freezing step (4) the suspension is frozen in liquid nitrogen to form pellets or is frozen on a tray.

14. The process according to any one of claims 10-13, further comprising washing the cells after harvesting in step (2).

15. The process according to any one of claims 10-14, wherein the lyoprotectants further comprise modified starch.