S. pombe active dry yeast and its use in the production of an alcoholic beverage

Abstract

The invention relates to a method for preparing dried Schizosaccharomyces, preferably S. pombe, yeast product, which product is an Active Dried Yeast (ADY), the method comprising - growing a viable Schizosaccharomyces, preferably S. pombe, in a culture medium, thereby obtaining a grown viable Schizosaccharomyces, preferably S. pombe, yeast having a protein content of 49.0 wt.% or less based on dry yeast weight; - recovering the grown Schizosaccharomyces, preferably S. pombe, yeast from the culture medium and obtaining a suspension of recovered viable Schizosaccharomyces, preferably S. pombe, cells; - drying the suspension of recovered Schizosaccharomyces, preferably S. pombe,, thereby obtaining the Active Dried Yeast, the Active Dried Yeast containing at least 1.0 x 1010 viable Schizosaccharomyces, preferably S. pombe, cells per gram Active Dried Yeast, and having a protein content of 49.0 wt.% or less based on dry weight of the Active Dried Yeast. The invention further relates to Schizosaccharomyces, preferably S. pombe, ADY and various uses thereof.

Description

[0001] P137414PC00

[0002] Title: S. pombe Active Dry Yeast and its use in the production of an alcoholic beverage

[0003] The invention relates to a method for obtaining a dried Schizosaccharomyces, preferably S. pombe, product, typically an Active Dried Yeast (ADY). The invention further relates to a dried Schizosaccharomyces, preferably S. pombe, product, typically an ADY. The invention further relates to the use of an ADY according to the invention in the production of an alcoholic beverage, in particular a distilled alcoholic beverage, such as rum.

[0004] Active Dried Yeast is a type of a dried yeast product, typically ready-to-use, only requiring unpackaging and rehydration. ADY’s are generally a granulated form of yeast, that maintains a high degree viable cells and fermentative performance such that it is able to be used in applications such as baking, brewing, distilling, wine or as probiotics. Known ADY’s typically essentially consist of Saccharomyces yeast cells (including some residual water) and optionally an emulsifier, which cells are dehydrated to prolong shelflife without needing refrigeration or freezing. Shelf-life of ADY’s at a temperature in the range of about 20-30 degrees C should typically be about one year or more. Upon rehydration in water (or an aqueous liquid which may contain nutrients) of the ADY, the yeast cells are rehydrated and can be used for fermentation, i.e. at least the major part of the yeast cells in the ADY should be viable in order to be an acceptable product; typically at least for use in the fermentation of sugar -containing fermentation media a viability of about 80 % or more is considered acceptable, with a viability of at least about 85 % being preferred.

[0005] Rum is a liquor made by fermenting sugarcane juice, sugarcane molasse, sugarcane syrup or another sugarcane by-product, containing fermentable carbohydrate, in the presence of yeast. The distillate is generally a clear liquid that is often aged in wooden (such as oak) vessels (casks). Generally, Saccharomyces cerevisiae is used as the primary yeast, although additional (secondary) yeasts may be used to impart a more distinctive flavour, in particular in artisanal applications. Examples of non- Saccharomyces yeasts that can be used include Pichia, Hansensiaspora, Trulaspore and Schizosaccharomyces pombe.A problem found with Schizosaccharomyces yeast, such as S. pombe, is a poor robustness in the fermentation process, and in particular a low viability has been found for dried S pombe. Schizosaccharomyces yeast, such as S. pombe, is distinct from much used yeasts such as Saccharomyces, amongst others in that is a fission yeast, whilst Saccharomyces is a budding yeasts. Saccharomyces yeasts are also known for their relatively low sensitivity to dehydration; they can be dried relatively easily, whilst a high viability is maintained, in contrast to a non-Saccharomyces fission yeast species, like S. pombe.

[0006] It has been attempted to enhance viability of dried S. pombe on a laboratory scale, by adding trehalose to a suspension of S. pombe before drying, see Roca-Domenech et al., Journal of Microbiology Research (2016), 6(4): 82-91. This paper illustrates the challenge of drying Schizosaccharomyces yeast (S. pombe) without losing most of its viability. The drying involved exposure to dry air at 28 degrees C for about 20 hours. No information is given about the extent of drying, i.e. details about the residual moisture content are lacking. Also, details about long-term stability (shelf-life) of the dried yeast are lacking. Viability after rehydration was generally less than 30 %, unless trehalose was added before drying. Addition of trehalose to the suspension resulted in an improved viability to up to about 50 % of its original viability before dehydration. The dried yeast in this paper is mentioned to be an active dried yeast (ADY). However, it is apparent that this term is used loosely. Active Dry Yeast as known in the art is generally a dried form of yeast, that maintains a high degree viable cells and fermentative performance such that it is able to be used in applications such as baking, brewing, distilling, wine or probiotics, also after storage at room temperature, in particular after at least about 2 years of storage at room temperature. As illustrated in Example 2, with the method of Roca-Domenech et al. total cell count directly after drying is well below 1 x 1010cells (Table 4), and a drastic loss in viability is observed already after 1 week of storage (Table 3). Further, in practice, in contrast with the dried yeast of Roca-Domenech, ADYs generally are in dried particulate form (such as granulate or noodle form) and generally essentially consists of yeast cells and an emulsifier, without further additives, such as sugars like trehalose.

[0007] However, commercial availability of Schizosaccharomyces products in brewing applications remains very limited. Commercial SchizosaccharomycesADY’s are not known to the inventors. ProMalic® is a commercial product which is composed of encapsulated Schizosaccharomyces in double-layered alginate beads, which has to be stored at 4-5 degrees C. Even then it has a relatively low shelf-life of 4 months, according to a technical datasheet available from AEB SPA, Brescia, Italy). Further, the production method of ProMalic® is unknown. ProMalic® is used in the production of wine at the beginning of the fermentation. It is used to reduce the acid content, by metabolising malic acid. The ProMalic® is removed once the malic acid content is sufficiently reduced, after which the ethanol production by sugar fermentation takes place by another yeast.

[0008] The inventors realised that it would be desirable to provide an Active Dried Schizosaccharomyces Yeast, in particular an Active Dried S. pombe Yeast, which can be used satisfactorily without being encapsulated, as in the fermentative production of an alcoholic beverage, in particular a spirit, such as a rum or another spirit made with sugar from sugar cane. This would allow use of Schizosaccharomyces, in particular S. pombe, on an industrial scale in a more robust manner and / or simplified manner. It would in particular be desirable to be able to provide such as dried product with conventional industrial equipment for the drying of yeasts, more in particular with a minimal need for additives to protect the yeast from the adverse effects of drying or rehydration. However, it has been found a challenge to process Schizosaccharomyces, such as S. pombe, into such dry products on an industrial scale with a satisfactory shelf-life and performance for industrial use in practice. Besides loss of viability, S. pombe has been found more heat-sensitive, leading to burning effects, during process steps wherein temperature rises to a temperature at which Saccharomyces yeasts can be processed satisfactorily. Also significant formation of hard lumps during the removal of water has been noticed. Significant lump formation is detrimental in that it can lead to clogging during processing. Besides, lumps in the yeast product are generally not appreciated by brewers who would be using the dried yeast product, as it can detrimentally affect rehydration of the yeast.

[0009] It is an object of the present invention to provide a dried Schizosaccharomyces product, in particular an S. pombe product, (a so called Active Dried Yeast) that can be produced on an industrial scale and has satisfactory viability for use in the fermentation of sugar-containing fermentation media, inparticular a mash, a wort or a most, to produce ethanol. In particular it is an object to provide a method for preparing a dried S. pombe product that can be carried out using industrial equipment known for the preparation of other active dried yeast products.

[0010] The inventors found that it is possible to dry Schizosaccharomyces, in particular S. pombe, using known equipment suitable for use on an industrial scale to obtain an Active Dried Yeast with satisfactory viability. The inventors found that this is possible without needing to add special protective additives, like trehalose, to the yeast suspension to be dried. The inventors realised that (i) controlling the protein level of the Schizosaccharomyces cells, in particular S. pombe cells, and optionally (ii) providing Schizosaccharomyces cells, in particular S. pombe cells, with a significant amount of a specific sugar makes it possible to provide an Active Dried Yeast having satisfactory properties, in particular viability, for use in brewing applications as an ethanol producing yeast.

[0011] Accordingly, the present invention relates to a method for obtaining a dried Schizosaccharomyces, preferably S. pombe, product, which product is typically an Active Dried Yeast, the method comprising

[0012] growing a viable Schizosaccharomyces, preferably S. pombe, in a culture medium, thereby obtaining a plurality of grown viable Schizosaccharomyces cells, preferably S. pombe cells, having a protein content of 49 wt.% or less based on dry weight of the cells and preferably a trehalose content of at least 10 wt.%, based on dry weight of the cells;

[0013] recovering the grown viable Schizosaccharomyces cells, preferably S. pombe cells, from the culture medium and obtaining a suspension of recovered viable Schizosaccharomyces, preferably S. pombe, cells;

[0014] drying the suspension of recovered viable Schizosaccharomyces cells, preferably S. pombe cells, thereby obtaining the Schizosaccharomyces product, preferably S. pombe product, which product typically is an ADY, the Schizosaccharomyces product, preferably S. pombe, product (typically Active Dried Yeast) containing at least 1.0 x 1010viable Schizosaccharomyces cells, preferably S. pombe cells, per gram Active Dried Yeast, the Active Dried Yeast having a protein content of 49 wt.% or lessbased on dry weight and preferably a trehalose content of at least 10 wt.% based on dry weight.

[0015] Further, the present invention relates to a Schizosaccharomyces product, preferably an S. pombe product, in particular an Active Dried Yeast product, preferably a product obtainable by a method according to the invention, comprising at least about 1 x 1010viable Schizosaccharomyces, preferably S. pombe, cells per gram dry weight, the Schizosaccharomyces, preferably S. pombe, product having

[0016] - a protein content of 49 wt.% or less, based on dry weight

[0017] -a trehalose content of at least 10 wt. %, based on dry weight.

[0018] Particularly good results in terms of viability, stability of viability during storage, fermentative capacity (ethanol production rate), have been achieved with S. pombe. An alternative advantageous Schizosaccharomyces in accordance with the present disclosure is S. Japonicus. Thus, in a specific advantageous embodiment, the ADY comprises Schizosaccharomyces Japonicus. Conditions, features etc described herein below for S. pombe, S. pombe ADY, the production thereof and the use thereof, may also be applied to S. Japonicus, S. Japonicus ADYs, the production thereof and the use thereof.

[0019] A further Schizosaccharomyces, preferably S. pombe, product in accordance with the present invention is an intermediate product obtained in a method of the invention, such as the grown S. pombe in the culture medium, a retentate of a filtration or centrifugation,a suspension, a cream yeast, an extrudate as described herein. Active Dried Yeast generally at least substantially consists of yeast material as the major component, typically a minor amount of residual moisture, and preferably a minor amount of emulsifier. The ADY is generally packaged during storage. Thus in an embodiment, the Active Dried yeast in accordance with the invention is in a packaging. The packaging may be any container suitable for storage of ADY’s. Suitable packaging is generally known in the art and include bags, jars, foils, pots etc.. Generally, the packaging is air-tight, moisture proof and lightblocking, which are well known qualities of packaging in the art. In particular advantageous is a foil vac pack, sealed under vacuum. The ADY is generally used, i.e. rehydrated and employed in its intended application, such as a fermentative rolein the production of an alcoholic beverage, without being surrounded by any protective material, such as an encapsulation or packaging.

[0020] It should be noted that the final dried product (the ADY) ready to be stored and subsequently used after rehydration, generally has a moisture content of 10 wt.% or less based on total weight; intermediate products obtained in a method for obtaining an ADY in accordance with the invention prior to a final drying step, such as a fermentate, a retentate, cream yeast or an extrudate usually have a moisture content of more than 10 wt.% based on total weight. The dried Schizosaccharomyces product, in particular the S. pombe product, generally has a content of Schizosaccharomyces material, preferably S. pombe material, of at least about 80 wt.% based on total weight, preferably at least about 85 wt.% based on total weight, more preferably at least 90 wt.% based on total weight. The Schizosaccharomyces material, preferably S. pombe material, includes the viable S. pombe (forming at least the bulk) at -if present- any non-viable Schizosaccharomyces or remains of Schizosaccharomyces (lysate, debris like cell wall components, and the like). The ADY in accordance with the invention can be stored whilst maintaining satisfactory viability for several months or years, usually for at least 12 months, preferably for about 24 months or more, in particular up to 48 months or up to about 36 months. In particular, such a shelf-life is possible without needing to maintain the temperature below 10 degrees C, i.e. without needing refrigeration to about 4 degrees C or storing the ADY in a freezer; more in particular, such shelf-life is feasible in a storage under ambient conditions. If desired the temperature may be stored at a maximum temperature e.g. of up to 30, up to 25 or up to 20 degrees C.

[0021] The Active Dry Yeast (obtained) in accordance with the invention in maintains a high degree viable cells and fermentative performance such that it is able to be used in applications such as baking, brewing, distilling, wine or probiotics. It can be stored for a long time, such as over a year, in particular 2-4 years whilst maintaining satisfactory properties for its intended use. As illustrated in the examples, the invention provides Schizosaccharomyces ADY products, in particular S. pombe ADY products, with over 85 % viability, a dry weight of 94 % and more, and having viable cell counts of over 2 x 1010viable cells per gram ADY. Total viable yeast count(per gram ADY) and % viability(% live cells on total cells)can be determined with commercially available yeast cell counting device and software, in particular making use of image analysis, such as an ABER Countstar (ABER COUNSTSTAR Yeast model 7600-00 instrument) using the corresponding Countstar method supplied with the instrument.

[0022] The ADY in accordance with the invention can be used in a wide variety of applications, in particular in the production of alcoholic beverages to produce alcohol from sugar and / or to contribute to a distinct flavour. Accordingly, the present invention further relates to a method for producing an alcoholic beverage, comprising

[0023] - providing an aqueous liquid comprising a fermentable carbohydrate and reconstituted viable Schizosaccharomyces cells, preferably S. pombe cells, which reconstituted (rehydrated) viable Schizosaccharomyces cells, preferably S. pombe cells, are cells of a rehydrated ADY according to the invention or cells of a rehydrated dried Schizosaccharomyces, preferably S. pombe, product obtained by a method according to the invention; and

[0024] - subjecting the aqueous liquid comprising the fermentable carbohydrate to fermentation by the rehydrated viable Schizosaccharomyces cells, preferably S. pombe cells, under formation of ethanol, wherein a fermentate is obtained.

[0025] In a specific embodiment, the invention provides thus a method for producing an non-alcoholic beverage, comprising

[0026] - providing an aqueous liquid comprising a fermentable carbohydrate and reconstituted viable Schizosaccharomyces cells, which rehydrated viable Schizosaccharomyces cells are cells of a rehydrated Active Dried Yeast according to the invention or cells of a rehydrated Active Dried Yeast obtained by a method according the invention;

[0027] - subjecting the aqueous liquid comprising the fermentable carbohydrate to fermentation by the rehydrated viable Schizosaccharomyces, wherein a fermentate is obtained, from which the non-alcoholic beverage is made. Advantageously, the Schizosaccharomyces cells in this embodiment are selected from S. japonicus cells and S. pombe cells.

[0028] The invention further relates to the use of the ADY according to the invention, in the production of an alcoholic beverage.The ADY according to the invention is advantageously used as a dry starter culture for use in the production of an alcoholic beverage.

[0029] The ADY according to the invention is particularly useful in the production of a distilled alcoholic beverage. A preferred use is in the fermentation of sugar from sugar cane, such as in the production of a rum. It is also possible to use the ADY according to the invention in the production of a non-distilled alcoholic beverage, such as a wine, a beer or a cider.

[0030] Besides using it for the production of alcohol, the ADY can also be used to reduce the malic acid content of a liquid, such as a fruit juice.

[0031] For the purpose of clarity and a concise description, features are described herein as part of the same or separate embodiments, however, it will be appreciated that the scope of the invention may include embodiments having combinations of all or some of the features described.

[0032] As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well.

[0033] The term "or" includes any and all combinations of one or more of the associated listed items, unless the context clearly indicates otherwise (e.g. if an “either....or” construction is used).

[0034] It will be understood that the terms "comprises" and "comprising" specify the presence of stated features but do not preclude the presence or addition of one or more other features. It will be further understood that when a particular step of a method is referred to as subsequent to another step, it can directly follow said other step or one or more intermediate steps may be carried out before carrying out the particular step, unless specified otherwise.

[0035] In the context of this disclosure, the term "about" means generally a deviation of 15 % or less from the given value, in particular a deviation of 10% or less, more in particular a deviation of 5% or less, even more in particular a deviation of 2% or less.

[0036] The term “substantial(ly)” or “essential(ly)” is generally used herein to indicate that it has the general character or function of that which is specified. When referring to a quantifiable feature, these terms are in particular used toindicate that it is for at least 80 %, more in particular at least 90 %, even more in particular at least 95 % of the indicated feature.

[0037] When referring to “essentially free”, this generally means that a component is absent (not detectable), or - if present - in such low amount that it does not cause any significant effect. In particular, a composition is considered essentially free of a component.

[0038] As is used herein, the term “viable”, refers to an organism capable of reproduction / colonisation at least when in non-dormant state. I.e. dried yeast cells (in a dormant state) are viable, if capable of reproduction / colonisation when rehydrated. Suitable rehydration liquids are generally known in the art.

[0039] The method for obtaining the Schizosaccharomyces ADY, preferably the S. pombe ADY, in accordance with the invention requires a growing step in a fermenter containing the culture medium, wherein the Schizosaccharomyces cells multiply. The culture medium contains a sugar, a nitrogen source and further nutrients needed for growth. The nutrients may be fed continuously or intermittently to the fermenter, containing the growing Schizosaccharomyces cells. Typically, the fermenter is aerated for optimal growth. The nutrients may be known as such for growing cells, albeit that they are advantageously selected and used in amounts favouring the growth of Schizosaccharomyces cells, preferably S. pombe cells, having a protein (and preferably a trehalose content) in accordance with the present invention. Typically, the nutrients include a carbon source (usually a sugar), a nitrogen source (such as amino acids, ammonium salt, urea), a phosphorus source (such as a phosphate salt), vitamins and minerals (such as magnesium, potassium, trace elements, like zinc and iron).

[0040] For controlling the protein content ( and - if desired - the trehalose content) in the Schizosaccharomyces cells, preferably the S. pombe cells, that are to be recovered from the fermenter, one has several options, including the choice of nutrients (qualitatively, quantitatively, or both). Particularly good results have been achieved with a medium containing glucose, more in particular a medium with sugar cane or sugar cane molasse as the carbohydrate source. Yeast extracts are advantageously used as a source for essential vitamins, nitrogen, minerals and growth factors. Ammonium salts are also advantageously used. Further knownsubstances for use in producing or storing yeast products can be used for production or for the inoculation medium containing Schizosaccharomyces, preferably the S. pombe, to be added to the fermenter wherein production of S. pombe takes place E.g. peptone, agar (such as GYP agar) can be present in the inoculation medium. In particular, good results have been achieved with a fermentation medium containing - in addition to water - molasse, such as beet molasse, cane molasse or both, as a carbon source, more preferably as essentially the sole carbon source or at least the major carbon source (providing >50 % of the carbon), the medium further containing ammonia (e.g. as ammonium hydroxide) as a nitrogen source, more preferably as essentially the sole nitrogen source or at least the major nitrogen source (providing> % of the nitrogen), the medium further containing phosphate (e.g. as phosphoric acid), sulphate, minerals (such as zinc, copper, magnesium, calcium), B-vitamins (such as pantothenate, thiamine, pyridoxine, biotin, niacin). In a specific embodiment, the fermentation medium essentially consists of said ingredients. Advantageously, an inactive yeast product is added as a nutrient; it plugs a number of different nutritional gaps all at the same time without adding in (large amounts of) expensive singular vitamins etc. This nutrient may then also provide a (minor) amount of the nitrogen and carbon, in particular in addition to the ammonia (N-source) and molasse (C-source).

[0041] The inventors found that growing Schizosaccharomyces yeast (such as S.pombe yeast) with known production methods may result in a protein content of up to about 60 wt.%, based on dry weight. The inventors realised that the high protein content is a reason why such cells are not processable in a satisfactory manner to obtain a satisfactory dried ADY. The inventors found that too high a protein content did not allow for a sufficient degree of drying, at least not whilst maintaining sufficient content of viable yeast to qualify as an Active Dried Yeast, as acceptable for industrial application.

[0042] The inventors realised not only that it is important to obtain Schizosaccharomyces, preferably an S. pombe, having a protein content within a certain range for obtaining the Schizosaccharomyces ADY, preferably the S. pombe ADY, they also realised that it is possible to aim at a target %protein by estimating how much dry biomass will be made and then calculate how much nitrogen needs to be added in order to achieve that target. Nitrogen sources can include one ormore selected from the group of ammonia, urea, ammonium phosphate (e.g. diammonium phosphate (DAP)), ammonium sulphate, amino acids and peptides.

[0043] The production of protein by the yeast cells can further for instance be reduced by nutrient limitation, in particular using a reduced amount of nitrogen source (relative to optimal growth conditions). It is also possible to choose to use a specific nitrogen source which has limited contribution to the protein production; e.g. the inclusion of an ammonium salt (such as ammonium sulphate). Another option is growing the cells at relatively low temperatures, which can suppress protein formation. One may also consider the use of a inhibitor of protein synthesis that interferes directly or indirectly with ribosomal function, without being highly toxic to the yeast cells. For instance one may consider a compound that targets the TOR pathway, e.g. Torinl or rapamycin. One may also consider targeted gene modification to downregulate genes involved in protein production. However, it is preferred to provide a non- GMO S. pombe product. During the production process, the skilled person may take one or more samples of the Schizosaccharomyces, such as the S. pombe, that is being produced, measure the protein content, and adjust the process if the protein content of the Schizosaccharomyces (such as S. pombe ) is outside a desired range.

[0044] During the production process, the skilled person may take one or more samples of the Schizosaccharomyces cells, preferably the S. pombe, that is being produced, measure the protein content, and adjust the process if the protein content of the Schizosaccharomyces is outside a desired range.

[0045] Exposing the yeast cells to a stress condition during the growing is also an effective option to stimulate trehalose production, e.g. using a heat shock or a high osmolarity. Overexpression of the TPS1 gene and of the 0TPS2 gene may also be considered. Nitrogen-source limitation and the use of an ammonium salt have also been found favourable for stimulating trehalose production. Thus, modifying conditions relating to the nitrogen source has been found particularly suitable to provide an Active Dried Schizosaccharomyces Yeast, preferably the S. pombe Active Dried Yeast, having a protein content and - if desired - a trehalose content in accordance with the invention with a high viability. The inventors found that nutrient limitation is an effective way to grow viable Schizosaccharomyces, such as the S pombe, in a satisfactory yield within a reasonable time.Particularly good results have been achieved with a method, wherein the yeast is grown whilst reducing the sugar feed rate below normal yeast demand while maintaining aeration. Without being bound by theory, the inventors think this “tricks” the yeast into expecting that there is a starvation event coming, and the yeast responds by accumulating trehalose as a storage carbohydrate to be used when times are tough in order to survive. In combination, or alternatively, oxygen feed rate (aeration) can be regulated (in particular below normal yeast demand) to stimulate the yeast to start accumulating this storage sugar.

[0046] During the production process, the skilled person may take one or more samples of the Schizosaccharomyces, such as the S. pombe, that is being produced, measure the trehalose content, and adjust the process if the trehalose content of the Schizosaccharomyces is outside a desired range. Thus, in a particularly preferred method the sugar (such as molasse) feed rate, the oxygen feed rate, or both are set or regulated at a rate wherein the grown Schizosaccharomyces, preferably S. pombe, yeast has a trehalose content of at least 10 wt.% based on dry weight, preferably of 12-25 wt.%, based on dry weight, more preferably at least 14 wt.% in particular at least 15 wt.% based on dry weight, more in particular at least 16 wt.% based on dry weight; and / or more preferably about 28 wt.% or less based on dry weight, more preferably 25 wt.% or less based on dry weight, in particular 25 wt.% or less based on dry weight, more in particular up to about 20 wt.% or up to about 18 wt.%, based on dry weight. The skilled person will know on the basis of the present disclosure and common general knowledge to adjust sugar feed rate, oxygen feed rate or both to bring respectively maintain the trehalose content of the grown yeast within a desired range.

[0047] Further, Schizosaccharomyces, such as S. pombe, growing conditions can be based on known conditions for the production of commonly used yeasts, e.g. Saccharomyces. E.g. temperature can be at about ambient temperature or an elevated temperature at which growing proceeds, e.g. in the range of about 20 to about 35 degrees C, in particular about 28 to about 34 degrees C.

[0048] It should be noted that a method according to the invention is generally carried out without adding a protein to the yeast product. The protein present in a Schizosaccharomyces product, preferably an S. pombe product, (made) according tothe invention (intermediate product or ADY) generally essentially consists of protein produced by the Schizosaccharomyces, preferably the S. pombe, in the product. I.e. the Schizosaccharomyces, preferably S. pombe, product (made) in accordance with the invention, such as the ADY, is generally essentially free of added protein. Generally the protein content in the Schizosaccharomyces, preferably S. pombe, product in accordance with the invention at least substantially consists of intracellular protein. Any trehalose which may be present in the Schizosaccharomyces product, preferably S. pombe product, such as the ADY, is generally also trehalose produced by the Schizosaccharomyces, such as the S. pombe. I.e. the product, such as the ADY is generally essentially free of added trehalose. It is possible that an ADY or other product of the invention contains a minor amount of trehalose that is extracellular due to excretion of protein or lysis of cells.

[0049] The protein content of the grown Schizosaccharomyces, preferably S. pombe, yeast in accordance with the invention, respectively of the ADY, is 49.0 wt.% or less based on dry weight of the cells. Too high a protein content has an adverse effect on the maximum level of drying of viable Schizosaccharomyces, preferably S. pombe. Too high a protein content or too low a protein content has an adverse effect on viability. Too high a protein content further contributes to burning effects at relatively low temperature and / or to lump fermentation during the further processing of the recovered cells in a dryer, such as an (commercially available) industrial dryer. A relatively low protein content may further have an adverse effect on the growing rate of the Schizosaccharomyces cells, preferably S. pombe cells, their fermentative activity, or both. Generally the protein content is more than 1 wt.% based on dry weight of cells that are grown respectively the ADY. In practice, the protein content of the yeast cells that are grown, respectively of the ADY, usually is at least 10 wt.%, based on dry weight, in particular at least about 15 wt.%, based on dry weight. Preferably, in particular in view of a desirable fermentative activity, growing rate, and / or viability, the protein content of the grown Schizosaccharomyces yeast, preferably S. pombe yeast, respectively of the ADY is about 18 wt.% or more based on dry weight or more, more preferably about 24 wt.% or more, based on dry weight, more preferably about 26 wt.% or more, based on dry weight, in particular about 28 wt.% or more, based on dry weight,more in particular about 29 wt.% or more based on dry weight, or about 31 wt.% or more based on dry weight. It is in particular surprising that a relatively low protein content in the yeast does not adversely affect viability and activity of the yeast to an unacceptable extent, since proteins are generally considered of key importance for healthy cells, in fact as illustrated in the Example a high viability is reachable at low protein content. A relatively high protein content may start to adversely affect the process of obtaining the ADY in one or more stages of producing the ADY, such as in a filtration, extrusion or drying after extrusion. Thus, in practice at least for advantageous de-watering, the protein content of the yeast cells that are grown, respectively of the ADY usually is about 45 wt.% or less, based on dry weight, in particular about 43 wt.% or less, based on dry weight. Preferably, the grown Schizosaccharomyces, preferably S. pombe yeast, respectively the ADY has a protein content of up to about 40 wt.% based on dry weight, more preferably of about 39 wt.% or less, based on dry weight, more preferably of about 38 wt.% or less, based on dry weight. In a specific embodiment, the ADY has a protein content of 37 wt.% or less, 35 wt.% or less, or 34 wt.% or less. In particular, good results are achieved with grown Schizosaccharomyces cells, preferably S. pombe cells, respectively an ADY having a protein content in the range of about 26 to about 40 wt.% based on dry weight, more in particular in the range of 26.6 to 38.1 wt.%, based on dry weight or in the range of 28 to 36 wt. %, based on dry weight. Such (S. pombe) cells respectively ADY allow in particular an advantageously low moisture content, good viability / high viable cell content per gram of yeast respectively ADY. Further, a protein content of about 26 to about 38 wt.% based on dry weight offers favourable production conditions, in particular with respect to growth rate, dewatering or both. The protein content can be determined using known methodology, in particular with a nitrogen analyser, such as (the Elementar Rapid Max N Exceed Nitrogen Analyser, using a multiplication factor of 6.25 of the measured nitrogen % to establish the protein content.

[0050] Satisfactory results are reachable with a trehalose content of less than 10 wt.% based on dry weight. The trehalose content of the grown Schizosaccharomyces yeast, preferably S. pombe yeast, respectively the ADY is preferably at least 10 wt.%, based on dry weight, more preferably at least 12 wt.%, more preferably at least 14 wt.% in particular at least 15 wt.% based on dryweight, more in particular at least 16 wt.% based on dry weight. Such a trehalose content can provide a positive effect on survivability during drying, a positive shelflife or a positive effect on viability after dehydration and rehydration. At a very high trehalose content, the gains in viability are limited. Generally, the trehalose content of the yeast respectively ADY is about 30 wt.% or less based on dry weight. Preferably, the trehalose content of the yeast respectively is about 28 wt.% or less based on dry weight, more preferably 25 wt.% or less based on dry weight, in particular 25 wt.% or less based on dry weight, more in particular up to about 20 wt.% or up to about 18 wt.%, based on dry weight. As follows from the above, the trehalose generally is Schizosaccharomyces trehalose, preferably S. pombe trehalose, i.e. at least substantially forming part of the yeast cells. ADY in accordance with the invention is generally essentially free of added trehalose. If any trehalose is present in an ADY that does not form part of the yeast cells, it is generally trehalose originating from the Schizosaccharomyces, such as theS, pombe, such as from any (no longer viable) cells that may have been damaged whereby the trehalose may have been released. Trehalose content (can be determined using known methodology, such as HPLC. In particular the trehalose content of the yeast is determinable by extracted the trehalose from the yeast cell with hot 96% ethanol and analysing the resulting liquid with HPLC.

[0051] Recovery of the grown Schizosaccharomyces yeast, preferably S. pombe yeast, from the culture medium can be based on a manner known per se, e.g. the recovery can involve centrifuging, filtration or both. The recovered yeast cells are usually washed with (process) water (such as tap water) to remove unwanted fermentation products and nutrient remains. The resultant aqueous yeast suspension may be referred to as a cream yeast. The cream yeast generally at least substantially consists of yeast and the aqueous phase (water). In accordance with the invention, the cream yeast generally has a yeast content of about 15 to about 25 wt.%, based on dry yeast weight, in particular of 17-22 wt.% based on dry yeast weight. The water content of the cream yeast is generally about 85 to about 75 wt.%, in particular 83-78 wt.%, based on total weight. The cream yeast is usually obtained without adding sugars, such as trehalose to the yeast cells or cream yeast.Preferably, the cream yeast obtained after centrifugation and washing (typically with water, such as tap water) is subjected to filtration to reduce the water content, thereby obtaining a retentate containing the grown viable Schizosaccharomyces, preferably S. pombe, cells. Alternatively, the filtration may be performed directly on the culture medium to obtain a retentate which is washed (typically with water, such as tap water) to obtain the cream yeast.

[0052] Filtration to recover the yeast from the culture medium or filtration of the cream yeast obtained after washing can be done in any way suitable for yeast cells. The protein content as described herein has been found important in this step too. As indicated above, too high a protein content may adversely affect the dewatering effectivity or efficiency. Particularly preferred is the use of a filter press or a vacuum filtration system, thereby obtaining a retentate containing the grown Schizosaccharomyces, preferably S. pombe, cells. In particular good results are achieved with a filter press. The retentate, which may also be referred to in the art as a dewatered yeast cake or crumble, generally has a yeast solids content of at least about 26 wt.% based on total weight; Generally the yeast solids content is up to about 34 wt.%. Preferably the yeast solids content of the retentate is about 30-32 wt. %.

[0053] It has been found particularly advantageous to subject the retentate (or - if desired - the cream yeast) to an extrusion step, rather than to directly feed it to a dryer. For extrusion, the slurry (such as the retentate or the cream yeast) to be extruded usually has a dry weight of at least about 26 wt.%, preferably at least 28 wt.% or at least 30 wt.% for an advantageous consistency. Usually, the maximum dry weight for the slurry to be subjected to extrusion is determined by the maximum that is still pumpable through the extruder, and may in particular be up to about 35 wt.%, preferably up to 34 wt.%, more preferably up to 32 wt.% For extrusion in particular, conventionally grown S. pombe (with a high protein content, with a low trehalose content) has been found unsatisfactory. However, due to the change in protein content and optionally trehalose content of the S. pombe cells, extrusion can successfully be employed, without undesired levels of browning or lump formation. The resultant extrudate offers an advantage in drying compared to directly drying the cream yeast or retentate, e.g. in terms of uniform drying, enhanced rehydration after drying or enhanced drying efficiency. Theuniform drying is in particular important to avoid relative wet spots in the dried product, which may adversely affect viability and / or shelf life.

[0054] The method of the invention advantageously includes the addition of an emulsifier at some point in the process before the final drying step. The emulsifier can be selected from known emulsifiers that are compatible with yeasts, in particular known emulsifiers for use in the production of ADY’s. Particularly useful emulsifiers include sorbitan monostearate, lecithin, polysorbate, monoglycerides and diglycerides. Usually, the emulsifier is added after recovering the cells from the culture medium wherein they have been grown. Preferably, the emulsifier is added to the cream yeast or after filtration to the retentate (crumble yeast). The presence of an emulsifier can be advantageous for the drying process. Further, an emulsifier can be advantageous to facilitate rehydration. In particular, good results have been achieved with sorbitan monostearate. A further preferred group of emulsifiers are Citrems (citric acid esters of mono- and diglycerides of fatty acids). Various Citrems are e.g. described in the 87thJECFA - Chemical and Technical Assessment (CTA), 2019 of the Food and Agriculture Organization of the United Nations, by Maria Frutos Fernandez. Usually, the emulsifier, preferably the sorbitan monostearate, is used in an amount of 0.1% to 1.5 wt.%, in particular of 0.4- 1.2 wt.%, based on dry weight. In a particularly advantageous embodiment, an emulsifier is added to the retentate (crumble) before extrusion.

[0055] Before final drying, the retentate, respectively the extrudate, is advantageously broken up. For this an (industrial) shaking conveyor is particularly preferred, although other means maybe employed.

[0056] Final drying to obtain the dried yeast product can be carried out in a manner known per se. However, in particular, good results have been achieved with a fluidised bed dryer.

[0057] It should be noted that a method according to the invention is generally carried out without needing to add a protective compound, such as a protective sugar, like trehalose. The trehalose present in a Schizosaccharomyces product, preferably an S. pombe product according to the invention (intermediate product or ADY) generally essentially consists of trehalose produced by the Schizosaccharomyces, such as the S. Pombe, in the product. If present, any extracellular trehalose generally originates from Schizosaccharomyces, preferablyS. pombe, used or formed in the production of the Schizosaccharomyces product, preferably the S. pombe product. I.e. the Schizosaccharomyces product, in particular the S. pombe product, (made) in accordance with the invention is generally essentially free of added trehalose.

[0058] The dried yeast product (the ADY) can be packaged, e.g. as a powder, a noodle shaped product or a granulate (e.g. crumbled or as obtained with fluidised bed drying) without needing further treatment. The ADY is typically nonencapsulated. If desired the dried S. pombe yeast is compressed to form a compressed ADY.

[0059] The method according to the invention has been found particularly suitable for obtaining an Schizosaccharomyces ADY, in particular an S. pombe ADY, with an Schizosaccharomyces viability, preferably S. pombe viability of 80 % or more, preferably of 85 wt.% or more, more preferably of 90 wt.% or more. In principle, essentially all of the Schizosaccharomyces, in particular S. pombe, of the ADY can be viable. In practice, the viability can be less than 100 %, in particular after a prolonged time of storage or if a product has been stored under conditions generally known not to be recommended for ADY storage. Thus, in practice viability usually is 99 % or less. In particular, in practice viability can be or become 97 % or less, more in particular 96 % or less, 95 % or less or 90 % or less. The method of the invention allows the production of a Schizosaccharomyces ADY, in particular an S. pombe ADY, comprising at least 1 x 1010viable Schizosaccharomyces (such as S. pombe ) cells / gram product. Preferably, the ADY comprises at least 1.1 x 1010viable Schizosaccharomyces (such as S. pombe) cells / gram product, more preferably at least 1.5 x 1010viable Schizosaccharomyces (such as S. pombe) cells / gram product,. In a highly preferred embodiment, the ADY comprises at least 2. Ox 1010viable Schizosaccharomyces cells (such as S. pombe cells) per gram product. The maximum number of viable cells is determined by the average weight of the cells. Generally, the Schizosaccharomyces content, in particular the S. pombe content, of the ADY is about 3.5 x 1010viable Schizosaccharomyces cells (such as S. pombe cells) / gram product or less, in particular about 3.0 x 1010viable Schizosaccharomyces cells (such as S. pombe cells) / gram product or less, more in particular 2.5 x 1010viable Schizosaccharomyces cells (such as S. pombe cells) / gram product or less. If desiredthe product may comprise less than 2.0 x 1010viable Schizosaccharomyces cells (such as S. pombe cells) / gram product, e.g. about 1.5 x 1010viable Schizosaccharomyces cells (such as S. pombe cells) / gram product or less.

[0060] The dried S. pombe product or other Schizosaccharomyces product, such as the ADY, in accordance with the invention generally comprises viable Schizosaccharomyces cells, preferably viable S. pombe cells, as the major component, typically forming at least 70 wt.%, preferably at least 80 wt.%, more preferably at least 85 wt.% or at least 90 wt.% of the total weight of the dried product. The viable Schizosaccharomyces cells, preferably the viable S. pombe cells generally have about the same protein content (and preferably about the same trehalose content) as given elsewhere herein for the viable grown cells recovered from the culture medium. Further, non-viable Schizosaccharomyces (such as S. pombe (which may be killed cells, cellular debris etc.) may be present as a minor component, usually forming less than 20 wt.%, preferably less than 15 wt.%, in particular less than 10 wt.% of the total weight of the product. In practice, the amount of non-viable Schizosaccharomyces material, such as non-viable S. pombe material, may be at least 0.1 wt.%, at least 1 wt.%, at least 3 wt.% or at least 5 wt.%, based on total weight.

[0061] Usually, at least 90 wt.%, based on total weight of the ADY, is biomass of Schizosaccharomyces, preferably S. pombe, (i.e. viable S. pombe cells and optionally any non-viable S. pombe cells or parts thereof). Preferably at least 92 wt.%, more preferably at least 93 wt.%, in particular at least 92 wt.% based on total weight of the ADY is biomass of Schizosaccharomyces, preferably S. pombe. The upper limit of said biomass is determined by the residual water content and optionally present further minor component(s), in particular an emulsifier, of which sorbitan monostearate is a preferred example The residual water content of the product usually is 10 wt.% or less, based on the total weight of the dried product. For an advantageous viability and in view of storage stability, the residual water content preferably is in the range of 1-9 wt.%, more preferably in the range of 2-8 wt.%, in particular in the range of 3-8 wt.% or 4-6 wt.%. If present, the emulsifier content, preferably the sorbitan monostearate content, usually is up to 2 wt.%, preferably 0.1% to 1.5 wt.%, in particular of 0.4-1.2 wt.% or 0.4-1.1 wt.%, based on total weight. The solids content of a dried S. pombe product according tothe invention is at least 90 wt.%, preferably of 92-98 wt.% based on total weight, more preferably 93-98 wt.% based on total weight, in particular 94-97 wt.% v, more in particular 94.5-96.5 wt.% based on total weight.

[0062] The Schizosaccharomyces, in particular the S. pombe, preferably has a (cellular) phosphorous content, calculated as P2O5, of 1.5 – 2.2 wt.%, based on dry weight. Too low a phosphorus content has been found to have a destabilising effect on the cell membranes, whereby viability is reduced.

[0063] In particular, the present invention provides a Schizosaccharomyces ADY, preferably an S. pombe ADY. In an advantageous embodiment of the invention, the ADY particles generally have a length in the range of about 1 to about 2 mm and a diameter in the range of about 0.5 to about 0.8 mm. In a particularly preferred embodiment, the ADY has a dry solids content of 94%w / w or more, a protein content in the range of 26-36 wt.% on dry basis, a trehalose content of 10wt% or more on dry basis and a phosphorous content, calculated as P2O5, of 1.5-2.5wt.% on dry basis. The live yeast count of the ADY is generally between 1.0E+10cfu / g to 2.0E+10cfu / g. The % viability is generally 80% or more live yeast cells. Such ADY advantageously have a shelf-life of 2-4 years when stored between 20-30 degrees C. The ADY generally does not need to be refrigerated at 2-5 degrees C to maintain satisfactory fermentative performance and viability.

[0064] The invention further relates to method for producing an alcoholic beverage, comprising

[0065] providing an aqueous liquid comprising a fermentable carbohydrate and reconstituted viable Schizosaccharomyces, preferably S. pombe, cells, which reconstituted viable Schizosaccharomyces, preferably S. pombe, cells are cells of a reconstituted dried Schizosaccharomyces, preferably S. pombe, product, such as an ADY, according to the invention or cells of a reconstituted dried Schizosaccharomyces, preferably S. pombe, product, such as an ADY, obtained by a method according to the invention; subjecting the aqueous liquid comprising the fermentable carbohydrate to fermentation by the reconstituted viable Schizosaccharomyces, preferably S. pombe, cells under formation of ethanol, wherein a fermentate is obtained.Usually, the ADY is reconstituted in water (e.g. clean tap water) in a manner known per se, and then added to the aqueous liquid. The aqueous liquid can be a liquid usually used for a particular kind of brewing, such as a mixture of water and sugar cane juice or sugar cane byproduct (e.g. for rum and other sugar cane based liquids), most (e.g. for wine) or a wort (for malted cereal based liquids).

[0066] The Schizosaccharomyces ADY, preferably the S. pombe ADY, in accordance with the invention is in particular preferred for fermenting juice of sugarcane, sugarcane syrup, sugarcane molasse or another sugar cane by-product, which is typically diluted with water (in a known manner), thereby forming a fermentate containing ethanol (e.g. about 5 to about 10 % ethanol). Besides converting sugar into ethanol, Schizosaccharomyces, in particular S. pombe, produces flavour / aroma components that contribute to a characteristic taste. E.g.

[0067] 2-ethyl-3-methyl butyric acid and esters like ethyl butyrate and ethyl hexanoate give rise to the sweetness and fruitiness.

[0068] The fermentate, such as the fermentate obtained from the sugar cane juice or by-product, can be distilled and, if desired, subjected to further treatment, such as aging, to obtain a spirit (distilled beverage). If desired, the distillate is blended, diluted or both. Distilling and further treatment can be based on known methods for a spirit of interest. Thus, in particular a spirit can be obtained selected from the group of rum, Cachaqa, Agricole, aguardients, aguardiente de cana and charanda, clairin, and blends of rum and another spirit, such as spirits known in German language as ‘Rum-Verchnitt’ (cut rum) and Inlanderrum (Domestic rum). The rum can be any type of rum, including white rum, dark / gold rum, spiced rum, overproofed rum or aged rum.

[0069] It is also possible to use the Schizosaccharomyces product, such as the S. pombe product or the S. japonicus product of the invention for the production of a non-distilled fermented beverage, e.g. a beer, a wine, a rice wine, or a cider. The non-distilled fermented beverage maybe alcoholic or non-alcoholic. E.g. the Schizosaccharomyces product of the invention, in particular an S. pombe ADY or an S. japonicus ADY may be used in the production of a fermented tea, a nonalcoholic fermented cereal-based drink etc. Processes for making such drinks, as such, are also generally known in the art. The Schizosaccharomyces product of theinvention may in particular advantageously be included to contribute to a distinct aroma, e.g. adding to aroma complexity, or mouthfeel.

[0070] Next, the invention will be illustrated by the following example.

[0071] Example 1

[0072] Fermentations were conducted using 2L working volume fermenters using food grade molasses as a substrate. Fermentations were of aerobic fed batch type where temperature, sugar feed, aeration, pH, nutrient additions were controlled or dosed in a step wise manner. Nitrogen additions were varied from no addition(Og N) to high dose(10.5g N) in order to observe the behaviour and effect on the S.pombe culture which included monitoring % protein and the culture’s subsequent ability to be dried and made into a typical commercial style ADY product.

[0073] At the end of the growing stage, the yeast was recovered from the growing medium. Recovery included centrifugation and washing with (tap) water, thereby obtaining a cream yeast. The cream yeast was subjected to filtration after which the retentate (crumble containing the yeast) was extruded into noodles. The noodles were subjected to a breaking up step on a conveyor belt, and if satisfactory size reduction was achieved, the broken up extrudate was further dried in a fluidised bed drier to obtain the ADY.

[0074] Results are shown in the following table (added nitrogen source increasing from 0 to 10.5 g N from #1 to ‘Comparison’).Table 1

[0075] product# gdw / %Protein ADY Viable Cell Count / g % Viability

[0076] gFS** dry wt solids % (Countstar) F

[0077] 0.061 26.6 96.9 85.7 2.4E+10

[0078] 2 0.063 27.6 94.5 95.5 3.0E+10 30 0.14 29.2 94.5 95.5 3.0E+10 4 0.332 31 96.4 90.6 2.7E+10 5 0.359 38.1 94.8 87.5 2.3E+10

[0079] Did not sufficiently dry to an extent needed comparison 0.445 49.1

[0080]

[0081] for a commercial ADY

[0082] *in addition to N sources in the molasse

[0083] **gram product dry weight per gram fermentable sugars

[0084] * two batches made and combined before drying

[0085] ** three batches made and combined before drying

[0086] The results show that the protein content of the produced S. pombe is of prime importance to obtain a satisfactory ADY. Above 49.0 %, the extruded yeast noodles were sticky, whereby they could not be broken up sufficiently for good further processing in the dryer. It is further in particular surprising that the present invention allows the production of ADY’s with an extremely high viable cell count of over 2 x 1010viable cells per gram.

[0087] With respect to varying the nitrogen source, it was found that each source was suitable for producing S. pombe ADY according to the invention without substantial variations in solids content, viability and viable cell count after drying. Regarding product #1, made without further addition of the nitrogen source, it is observed that the molasse contained sufficient nitrogen source to have growing conditions, albeit at a relatively low yield.

[0088] Determination of dry weight, protein, and viability:% Dry weight: samples are weighed into a pre-dried metal tin and dried using a fan forced drying oven set at 105C for 18-24h. The samples are weighed again after drying in the oven with the weight loss calculated and recorded. This weight loss is the total water content that was in the original sample. Expressed as % dry weight or % dry solids.

[0089] % Protein(dry basis): samples were tested using the Elementar Rapid Max N Exceed Nitrogen Analyser and using the corresponding method that was supplied with the machine. Once the % nitrogen level is established this figure is multiplied by 6.25 to establish the protein level, this level then corrected for dry weight and expressed as % protein (dry weight basis).

[0090] Total viable yeast count(per gram ADY) and % viability (% live cells on total cells): samples were tested using the ABER Countstar Yeast model 7600-00 instrument and using the corresponding Countstar method that was supplied with the machine.

[0091] Example 2

[0092] The production of an ADY in accordance with the invention using the same S.pombe strain (internal reference: Y2009) as in Example 1 was compared with the drying of the same strain using the method of Roca-Domenech et al. (cited above, RD in Tables below). Further, a sample of a strain used in Roca-Domenech et al. (deposit nr. CECT 11197, obtained from Collection Espanola Cultivos Tipo(CECT)-Universitat de Valencia) was subjected to the method of of Roca-Domenech et al.

[0093] For the growing and drying of the method of Roca-Domenech et al, the following protocol was used:

[0094] Prepare 250g EMM medium as per instructions on packaging using sterile water. Aliquot 100g of media into sterile 250mL flask, inoculate one loop from each slope (either Y2009 or CECT11197 (Y2442)) to each flask and incubate at 32 degree C at lOOrpm for 24 hours.

[0095] Cool flasks on ice for one hour before spinning culture down in centrifuge at 4500rpm for 10 mins. Resuspend one half of each cell culture with pure water, and the other half of each cell culture with 5mL 10% w / v trehalose solution.Perform a cell count on each cell culture using haemocytometer.

[0096] Adjust cell culture to 5 x 107cfu / mL using respective resuspension solutions.

[0097] Dispense ImL of each culture into every well of two 12-well plates and desiccate uncovered in the presence of a dehumidifier set at 28 degree C for 24 hours.

[0098] After 24 hours, resuspend each well of the plates with ImL of resuspension solution (5mM MgSO4 for Trehalose dried samples and filtered pure water for water dried samples) by shaking at 150rpm and 37degree C for 5 minutes.

[0099] Once resuspended, pool together the wells of each respective condition to get enough volume and cells for testing.

[0100] Retain the remaining plate with dry cells with lid on and vacuum sealed in a polyethylene sleave for testing at 0, 7 and 14 days

[0101] Perform % viability and total viable yeast count using a haemocytometer and methylene blue stain.

[0102] For standard rum fermentation test, the protocol is:

[0103] Prepare 33%w / w Harwood Cane Molasses with 0.55g / L Diammonium phosphate, 0.15g / L nutrient ( inactive dried yeast)

[0104] Add 5mL of media into 50mL falcon tubes with pin hole in lid for ventilation Inoculate with ImL of each dried yeast suspension and begin fermentation at 33°C 130rpm for 48hrs

[0105] Take Brix% and HPLC samples at 24hrs and 48hrs

[0106] For the production of the ADY in accordance with the invention, the method was based on Example 1 with the following fermentation medium.Table 2: Fermentation medium:

[0107] when made up (full fermenter basis):

[0108] Ingredient Amount Units Beet Molasses 190 G

[0109] Cane Molasses 20 g

[0110] 25% Ammonium Hydroxide 5.7 G

[0111] 85% Phosphoric Acid 1.34 G Thiamine HC1 0.015 G Pyridoxine HC1 0.012 G Calcium Pantothenate 0.010 G

[0112] d-biotin 0.005 g

[0113] Niacin 0.013 g MgSO4-7H2O 0.8 g

[0114] ZnSO4 - 7H2O 0.2 g

[0115] CuSO4 - 5H2O 0.03 g

[0116]

[0117] Viability and total yeast count of the dried strains was determined immediately after drying, after 7 days of storage and after 14 days of storage. Storage was at 25 degrees C. Results are shown in Table 3 respectively Table 4.

[0118] Table 3 Experimental summary % viability 0,7,14 days, at 25 degrees C S.pombe Method Additives Rehydration 0 7 14 Strain during solution days days days drying

[0119] Y2009 Invention Pure Pure Water

[0120] 89.6* 89.2* 91.3* Water

[0121] Y2009 RD Pure Pure Water

[0122] 6.8 c a i#7.6 ( m a#0.60 o p r t ve) water

[0123] Y2009 RD 10% 5mM MgSO4

[0124] (comparative) solution 70.6 47.7 7.2

[0125] Trehalose

[0126] CECT RD Pure Pure Water

[0127] 22.5 13.7 2.1 11197A(comparative) water

[0128] CECT RD 10% 5mM MgSO4

[0129] 11197A(comparative) solution 78.9 38.5 5.0

[0130]

[0131] Trehalose* within SD

[0132] #within SD

[0133] Table 4: Experimental summary total yeast count(viable cells / gram) 0,7,14 days at 25 degrees C _

[0134] S.pombe Method Additives Rehydration 0 days 7 days 14 days Strain during solution

[0135] drying

[0136] Y2009 Invention Pure Pure Water

[0137] 2.3E+10 2.6E+10 2.4E+10 Water

[0138] Y2009 RD Pure Pure Water

[0139] 2.6E+08 2.2E+08 3.5E+07 (comparative) water

[0140] Y2009 RD 10% 5mM MgSO4

[0141] (comparative) solution 3.2E+09 1.9E+09 5.1E+08

[0142] Trehalose

[0143] CECT RD Pure Pure Water

[0144] 1.9E+09 1.7E+09 9.2E+07 11197A(comparative) water

[0145] CECT RD 10% 5mM MgSO4

[0146] 11197A(comparative) solution 4.4E+09 2.4E+09 3.1E+08

[0147]

[0148] Trehalose

[0149] In conclusion, for strain Y2009 the method of Roca-Domenech et al results in up to only 70.6 % viability after 0 days and shows a strong decline over the 14 days period to 7.2 % viability (drying in presence of trehalose, rehydration with magnesium sulphate) and less than 10 % viability when drying and rehydrating in water, whilst the method of the invention provides an essentially constant viability of about 90 % throughout the test period of 2 weeks for the same strain. This essentially constant viability also indicative of a desirably long shelflife of the ADY.

[0150] It is further interesting to note that changing the strain for the strain of Roca-Domenech et al in the method of the method Roca-Domenech et al also resulted in a lower viability, well below 90 % and suffered from a strong loss of viability during storage (from 78.9% to 5.0 % or from 22.5 % to 2.1 %, depending on drying and rehydration conditions).

[0151] Moreover, the results in Table 4 illustrate that the cell count in the ADY of the invention is well above 1.0 x 1010viable cells / ml throughout the test period, and that the method of Roca-Domenech et al results in much lower cell count than 1.0 x 1010viable cells / ml (highest was 4.4 x 109after 0 days for CECT 11197, dried in the presence of trehalose and rehydrated in magnesium sulphatesolution, which has a significant loss of viable cells by over a factor 10 after 2 weeks).

[0152] Table 5 shows a comparison of dried yeast chemistry results with dried yeast produced according to invention vs method as described in Roca-Domenech et al 2016

[0153] Table 5

[0154] Test Y2009 ADYAY2009* CECT 11197(Y2442)* % yeast solids g

[0155] dw / g 94.08 46.16 36.13 % protein g / g dw 30.46 42.45 38.14

[0156]

[0157] % trehalose g / g dw 18.40# 7.01 8.16

[0158] Adried yeast produced according to invention

[0159] * dried yeast produced according to the method as described in Roca-Domenech et al 2016 using EMM media

[0160] # trehalose produced by the yeast

[0161] Fermentation Results- dried yeast kept for 0 days @25C

[0162] The results below indicate the superior fermentation performance of the ADY produced according the invention The ADY was much faster in the first 24h indicating superior fermentations kinetics and shorter lag phase.

[0163] In the tables below:

[0164] HADY= High Activity Dried Yeast according to the invention

[0165] W= dried and rehydrated with water only

[0166] T= dried in 10% trehalose solution and rehydrated with 5mM MgSO4

[0167] Fermentation Results- dried yeast kept for 0 days @25 degrees CTable 6:

[0168] HPLC results in %w / v Oh

[0169] Flask code t=0 Wort

[0170] Harwood

[0171] Cane

[0172] Glucose 10.458

[0173] Fructose 8.649

[0174] Total sugars 19.107

[0175] Ethanol %v / v 0.000

[0176]

[0177] Table 7: HPLC results in %w / v after 24h fermentation; average between triplicates

[0178] Flask code Y2009 Y2009 Y2009 T CECT1119 CECT1119

[0179] HADY W 7 (Y2442) 7 (Y2442) T W

[0180] Glucose 2.149 4.901 3.342 4.541 3.409 Fructose 3.180 4.900 3.799 5.808 4.795 Total sugars 5.328 9.801 7.140 10.348 8.204 Ethanol %v / v 6.319 3.583 5.190 3.377 4.572

[0181] Stdev between triplicates

[0182] Flask code Y2009 Y2009 Y2009 T CECT1119 CECT1119

[0183] HADY W 7 (Y2442) 7 (Y2442) T W

[0184] Glucose 0.109 0.055 0.074 0.060 0.024 Fructose 0.015 0.081 0.023 0.052 0.107 Total sugars 0.124 0.136 0.097 0.112 0.131 Ethanol %v / v 0.069 0.033 0.010 0.026 0.107

[0185]

[0186] Table 8: HPLC results in %w / v after 48h fermentation. Average between triplicates

[0187] Flask code Y2009 Y2009 Y2009 T CECT1119 CECT1119

[0188] HADY W 7 (Y2442) 7 (Y2442) T W

[0189] Glucose 0.123 0.354 0.125 0.114 0.122 Fructose 0.000 1.834 0.000 0.000 0.000 Total Sugars 0.123 2.189 0.125 0.114 0.122 Ethanol %v / v 8.929 8.312 8.967 8.990 8.985

[0190] Stdev between triplicates

[0191] Flask code Y2009 Y2009 Y2009 T CECT1119 CECT1119

[0192] HADY W 7 (Y2442) 7 (Y2442) T W

[0193] Glucose 0.004 0.020 0.006 0.002 0.004 Fructose 0.000 0.061 0.000 0.000 0.000 Total Sugars 0.004 0.081 0.006 0.002 0.004 Ethanol %v / v 0.007 0.031 0.027 0.020 0.003

[0194]

[0195] Table 9: Brix % Average between triplicates

[0196] Flask code Y2009 Y2009 Y2009 T CECT1119 CECT1119

[0197] HADY W 7 (Y2442) 7 (Y2442) T W

[0198] Initial 22.2 22.2 22.2 22.2 22.2 24h 15.9 18.6 18.2 19.0 19.1 48h 12.9 13.6 14.1 12.9 14.1

[0199] Stdev between triplicates

[0200] Flask code Y2009 Y2009 Y2009 T CECT1119 CECT1119

[0201] HADY W 7 (Y2442) 7 (Y2442) T W

[0202] Initial 0.0 0.0 0.0 0.0 0.0 24h 0.1 0.1 0.0 0.1 0.4 48h 0.1 0.1 0.1 0.1 0.0

[0203]

[0204] Fermentation Results- dried yeast kept for 14 days @25 degrees C

[0205] Table 10

[0206] HPLC results in %w / v Oh

[0207] Flask code t=0 Wort

[0208] Harwood

[0209] Cane

[0210] Glucose 10.257

[0211] Fructose 8.761

[0212] Total sugars 19.018

[0213] Ethanol %v / v 0.000

[0214]

[0215] Table 11: HPLC results in %w / v., after 24h fermentation

[0216] Average between triplicates

[0217] Flask code Y2009 Y2009 Y2009 CECT11197 CECT11197

[0218] HADY W T (Y2442) W (Y2442) T Glucose 1.508 7.846 6.161 8.042 7.841 Fructose 2.666 6.958 5.909 7.923 7.614 Total sugars 4.174 14.804 12.070 15.965 15.455 Ethanol %v / v 6.695 0.451 1.015 0.061 0.206

[0219] Stdev between

[0220] triplicates

[0221] Flask code Y2009 Y2009 Y2009 CECT11197 CECT11197

[0222] HADY W T (Y2442) W (Y2442) T Glucose 0.062 0.041 0.081 0.022 0.028 Fructose 0.044 0.027 0.122 0.011 0.023 Total sugars 0.105 0.067 0.203 0.033 0.051 Ethanol %v / v 0.107 0.015 0.107 0.000 0.006

[0223]

[0224] Table 12: HPLC results in %w / v, after 48h fermentation. Average between triplicates

[0225] Flask code Y2009 Y2009 Y2009 T CECT11197 CECT11197

[0226] HADY W (Y2442) W (Y2442) T Glucose 0.116 0.818 0.294 1.183 0.875 Fructose 0.000 2.297 1.640 3.782 1.804 Total Sugars 0.116 3.115 1.934 4.965 2.679 Ethanol %v / v 8.838 7.500 8.327 5.800 7.782

[0227] Stdev between triplicates

[0228] Flask code Y2009 Y2009 Y2009 T CECT11197 CECT11197

[0229] HADY W (Y2442) W (Y2442) T Glucose 0.005 0.036 0.056 0.003 0.009 Fructose 0.000 0.029 0.089 0.009 0.018 Total Sugars 0.005 0.065 0.145 0.012 0.027 Ethanol %v / v 0.032 0.035 0.081 0.023 0.010

[0230]

[0231] Table 13 Brix %, Average between triplicates

[0232] Flask code Y2009 Y2009 Y2009 CECT11197 CECT11197

[0233] HADY W T (Y2442) W (Y2442) T Initial 22.2 22.2 22.2 22.2 22.2 24h 15.3 21.9 20.3 22.8 22.7 48h 12.8 14.0 13.8 15.8 13.7

[0234] Stdev between triplicates

[0235] Flask code Y2009 Y2009 Y2009 CECT11197 CECT11197

[0236] HADY W T (Y2442) W (Y2442) T Initial 0.0 0.0 0.0 0.0 0.0

[0237] 24h 0.1 0.1 0.2 0.0 0.1

[0238] 48h 0.1 0.1 0.1 0.2 0.1

[0239]

Claims

Claims1. Method for preparing a dried Schizosaccharomyces yeast product, preferably an S. pombe yeast product, which yeast product is an Active Dried Yeast, the method comprisinggrowing a viable Schizosaccharomyces, preferably S. pombe, in a culture medium, thereby obtaining a grown viable Schizosaccharomyces yeast preferably S. pombe yeast, the yeast having a protein content of 49.0 wt.% or less based on dry yeast weight;recovering the grown Schizosaccharomyces yeast, preferably S. pombe yeast, from the culture medium and obtaining a suspension of recovered viable Schizosaccharomyces cells, preferably S. pombe cells;drying the suspension of recovered Schizosaccharomyces yeast preferably S. pombe yeast, thereby obtaining the Active Dried Yeast, the Active Dried Yeast containing at least 1.0 x 1010viable Schizosaccharomyces cells, preferably S. pombe cells, per gram Active Dried Yeast, and having a protein content of 49.0 wt.% or less based on dry weight of the Active Dried Yeast.

2. Method according to claim 1, wherein the grown Schizosaccharomyces, preferably S. pombe, yeast has a trehalose content of at least 10 wt.% based on dry weight, preferably of 12-25 wt.%, based on dry weight.

3. Method according to claim 1 or 2, wherein the grown Schizosaccharomyces, preferably S. pombe, yeast has a protein content of 15- 45.0 wt.%, based on dry weight, preferably 18-43 wt.%, based on dry weight, more preferably 26-39 wt.%, based on dry weight.

4. Method according to claim 3, wherein the grown Schizosaccharomyces, preferably S. pombe, yeast has a protein content of less than 38 wt.%, in particular of 37 wt.% or less, more in particular of 36 wt.% or less.

5. Method according to any of the claims 1, 2, 3 or 4, wherein the recovery comprises subjecting the culture medium containing the grown Schizosaccharomyces, preferably S. pombe, yeast to filtration or wherein therecovery comprises centrifugation of the culture medium containing the grown Schizosaccharomyces, preferably S. pombe, yeast, which centrifugation is followed by filtration, thereby obtaining the suspension containing the grown viable Schizosaccharomyces, preferably S. pombe, cells.

6. Method according to claim 5, wherein the suspension has a dry weight of at least 26 wt.%, preferably of 28-32 wt.%, and the suspension is subjected to extrusion, thereby obtaining an extrudate comprising the grown Schizosaccharomyces, preferably S. pombe, cells, and drying the extrudate thereby obtaining the Active Dried Yeast.

7. Method according to any of the preceding claims, wherein the recovered yeast cells are washed with water, thereby forming a cream yeast of yeast cells suspended in water, the cream yeast having a total yeast content (based on dry yeast weight) of about 15 to about 25 wt.% of the total weight of the suspension, in particular of 17-22 wt.%, and a water content of about 85 to about 75 wt.%, in particular 83-78 wt.%, based on total weight of the suspension.

8. Method according to any of the preceding claims, wherein the suspension of recovered Schizosaccharomyces, preferably S. pombe, cells, e.g. the cream yeast, that is dried is essentially free of added trehalose, preferably free of added sugars.

9. Method according to any of the preceding claims, wherein the suspension contains an emulsifier, in particular sorbitan monostearate, preferably in an amount of 0.1% to 1.5 wt.%, in particular of 0.4-1.2 wt.%, based on dry weight.

10. Method according to any of the preceding claims, wherein the Active Dried Yeast has a trehalose content of 10-25 wt.% based on dry weight, a protein content of 26-38 wt.%, based on dry weight, a residual water content of at most 10 wt.% of the total weight, and wherein the viable S. pombe cells content is in the range of 1.5 x 1010to 3 x 1010S. cells / gram Active Dry Yeast.

11. Method according to any of the preceding claims, wherein the Schizosaccharomyces is S. pombe.

12. Method according to any of the claims 1-10, wherein the Schizosaccharomyces is S. japonicus.

13. Active Dried Yeast, the Active Dried Yeast having- at least 1 x 1010viable Schizosaccharomyces cells, preferably S. pombe cells, per gram Active Dried Yeast,- a protein content of 49.0 wt.% or less, based on dry weight.

14. Active Dried Yeast according to claim 13, having a solids content of 92- 98 wt.% based on total weight, preferably 93-98 wt.% based on total weight, more preferably 94-97 wt.% based on total weight.

15. Active Dried Yeast according to claim 13 or 14, wherein at least 90 wt.%, based on dry weight of the Active Dried Yeast is biomass of Schizosaccharomyces cells, preferably biomass of S. pombe cells, i.e. viable Schizosaccharomyces cells, preferably S. pombe cells, and optionally any non-viable Schizosaccharomyces, preferably S. pombe, cells or parts thereof.

16. Active Dried Yeast according to claim 15, wherein at least 95 wt.%, based on dry weight, preferably at least 97 wt.% of the Active Dried Yeast is biomass of Schizosaccharomyces, preferably of S. pombe, (i.e. viable Schizosaccharomyces, preferably S. pombe, cells and optionally any non-viable Schizosaccharomyces, preferably S. pombe, cells or parts thereof).

17. Active Dried Yeast according to any of the claims 13- 16, wherein at least 85 wt.% of the weight of the Active Dried Yeast is the weight of the viable Schizosaccharomyces cells, preferably S. pombe cells, as determined by haemocytometry with methylene blue.

18. Active Dried Yeast according to any of the claims 13-17, wherein the protein content is in the range of 15 to 45 wt.% based on dry weight, preferably18 to 40 wt.% based on dry weight, more preferably 26 to 39 wt.%, based on dry weight of the Active Dried Yeast.

19. Active dried yeast according to claim 18, wherein the protein content is less than 38 wt.%, in particular 37 wt.% or less, more in particular 36 wt.% or less, based on dry weight of the Active Dried Yeast.

20. Active Dried Yeast according to any of the claims 13-19, having a trehalose content of at least 10 wt.%, based on dry weight, preferably in the range of 10-25 wt. %, based on dry weight, more preferably of 12-20 wt. % based on dry weight.

21. Active dried yeast according to any of the claims 13-20, wherein the Active Dried Yeast is free of added protective compounds.

22. Active Dried Yeast according to any of the claims 13-21, having a phosphorous content, calculated as P2O5, of 1.5-2.5 wt.%, preferably 1.5 - 2.2 wt.%, based on dry weight.

23. Active Dried Yeast according to any of the claims 13-22, comprising sorbitan monostearate, preferably in a content of 0.1% to 1.5 wt.%, in particular of 0.4- 1.2 wt.%, based on dry weight.

24. Active Dried Yeast according to any of the claims 13-23, wherein the Active Dried Yeast is a powder, a granulate, a compressed product or a crumble product, the Active Dried Yeast having a solids content of at least 93 wt.%, preferably of at least 94 wt.%.

25. Active Dried Yeast according to claim 24, wherein the Active Dried Yeast at least substantially consists of granules having a length in the range of about 1 to about 2 mm and a diameter in the range of about 0.5 to about 0.8 mm.

26. Active dried yeast according to any of the claims 13-25, essentially consisting of Schizosaccharomyces, preferably S. pombe, one or more emulsifiers, and having a residual water content of up to 10 wt.%, preferably up to 7 wt.%.

27. Active dried yeast according to any of the claims 13-26, wherein the active dried yeast is a product obtainable by a method according to any of the claims 1- 12.

28. Active dried yeast according to any of the claims 13-27, wherein the Schizosaccharomyces is S. pombe.

29. Active dried yeast according to any of the claims 13-27, wherein the Schizosaccharomyces comprises S. japonicus.

30. Method for producing an alcoholic beverage, comprising- providing an aqueous liquid comprising a fermentable carbohydrate and reconstituted viable Schizosaccharomyces, preferably S. pombe, cells, which rehydrated viable Schizosaccharomyces, preferably S. pombe, cells are cells of a rehydrated Active Dried Yeast according to any of the claims 13-29 or cells of a rehydrated Active Dried Yeast obtained by a method according to any of the claims 1-12;- subjecting the aqueous liquid comprising the fermentable carbohydrate to fermentation by the rehydrated viable Schizosaccharomyces, preferably S. pombe, cells under formation of ethanol, wherein a fermentate is obtained, from which the alcoholic beverage is made.

31. Method according to claim 30, wherein the Schizosaccharomyces, preferably S. pombe, cells are used to ferment juice of sugar cane, sugarcane syrup, sugarcane molasse or another sugar cane by-product, and wherein the fermentate is subjected to distillation, under formation of a distillate enriched in ethanol.

32. Method according to claim 31, wherein the alcoholic beverage is a rum, cachaca or rhum Agricole.

33. Method according to claim 32, wherein the alcoholic beverage is a beer, a wine or a cider.

34. Method for producing an non-alcoholic beverage, comprising- providing an aqueous liquid comprising a fermentable carbohydrate and reconstituted viable Schizosaccharomyces cells, which rehydrated viable Schizosaccharomyces cells are cells of a rehydrated Active Dried Yeast according to any of the claims 13-29 or cells of a rehydrated Active Dried Yeast obtained by a method according to any of the claims 1-12;- subjecting the aqueous liquid comprising the fermentable carbohydrate to fermentation by the rehydrated viable Schizosaccharomyces, wherein a fermentate is obtained, from which the non-alcoholic beverage is made.

35. Method according to claim 34, wherein the Schizosaccharomyces cells are selected from S. japonicus cells and S. pombe cells.

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