Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
  • C12G1/00—Preparation of wine or sparkling wine
  • C12G1/02—Preparation of must from grapes; Must treatment and fermentation
  • C12G1/0203—Preparation of must from grapes; Must treatment and fermentation by microbiological or enzymatic treatment
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/14—Fungi; Culture media therefor
  • C12N1/16—Yeasts; Culture media therefor
  • C12N1/18—Baker's yeast; Brewer's yeast

Definitions

• the present invention relates to yeast compositions, in particular yeast starter cultures capable of fermenting carbohydrate sources.
• the compositions can comprise one or more yeast organisms belonging to the family of Saccharomycetaceae, preferably selected from the geni of Torulaspora and/or Kluyveromyces and excluding the genus of Saccharomyces, optionally in combination with one or more yeast organisms belonging to the genus of Saccharomyces.
• the compositions are preferably freeze dried or spray/fluid bed dried.
• the invention also pertains to uses of the compositions for fermenting a carbohydrate source and producing an edible or drinkable product.
• Grape juice is a very specialised ecosystem wherein only a few yeast species sur- make and proliferate.
• Examples of such few yeast species include the wild yeast families Brettanomyces, Debaromyces, Hanseniaspora, Kloeckera, Torulaspora Zygo- Saccharomyces and Kluyveromyces (Bauer & Pretorius: S.Afr. J. Enol. Vitic. Vol. 21 , special issue 2000, pp. 27-51). Survival and proliferation may be either desirable or undesirable depending on the yeast species in question.
• grape juice is an oxygen limited and acidic environment with a high concentration of carbohydrates, these conditions naturally favour some yeasts species, which will tend to outgrow yeast species having different requirements for optimal growth.
• the use of starter cultures that contain a mixture of selected yeast species may be considered for a person skilled in the art.
• One option for a person skilled in the art to obtain a desired ecosystem, which meets the desire of enhancing the growth of those yeast species that have desirable characteristics in terms of aroma and flavour, would be to compile these desirable yeast species that are able to outgrow the less desirable yeast species and let them make up the desired ecosystem.
• This ecosystem would be further improvable by addition of sulfur dioxide in antimicrobially effective amounts in order to inhibit or suppress the indigenous population of e.g. spoilage microorganisms.
• Saccharomyces cerevisiae is capable of carrying out a final ethanol production due to its extreme adaptability to low oxygen concentration and high ethanol concentration, while other yeast species which may occur in combination with S. cerevisiae, only grow in a few days before they are outgrown by S. cerevisiae. Consequently, such other yeast species can only be found in young wine and they do not contrib- ute to the fermentation process for more than a few days.
• Hansen et al. J.
• non-Saccharomyces yeast species such as Torulaspora delbrueckii and Kluyveromyces thermotolerans are examples of yeast species, whose ability to grow is very sensitive to low oxygen contents of the growth medium. Such species are therefore not capable of participating for a sufficiently long time in a fermentation wherein S. cerevisiae species are also taking part.
• the background for wishing to use mixed yeast starter cultures may be a desire to produce a wine having a more refined aroma or flavour.
• spontaneous fermentations in some cases may be more desirable than fermentations based on S. cerevisiae starter cultures
• spontaneous fermentations are difficult to control and therefore wine with different and unknown characteristics from batch to batch result.
• many wine makers today rely on industrially produced S. cerevisiae starter cultures, which unfortunately are devoid of those yeast species which are capable of generating other desirable aroma and flavours.
• the present invention comprises the aim of solving the problem of how to provide a starter culture, including a mixed starter culture, capable of fermenting e.g. grape juice without losing the desirable characteristics of less competitive non-
• Saccharomyces yeast species that grow vigorously early on in the fermentation process.
• a starter culture as a composition of cultures comprising at least one first yeast organism selected from the family of Saccharomycetaceae excluding the genus of Saccharomyces.
• composition comprising at least one first yeast organism selected from the family of Saccharomycetaceae excluding the genus of Saccharomyces and at least one second yeast organism selected from the family of Saccharomycetaceae excluding the genus of Saccharomyces.
• composition comprising at least one first yeast organism selected from the family of Saccharomycetaceae excluding the genus of Saccharomyces and at least one further yeast organism selected from the genus of Saccharomyces.
• a composition comprising at least one first yeast organism selected from the family of Saccharomycetaceae excluding the genus of Saccharomyces and at least one second yeast organism selected from the family of Saccharomycetaceae excluding the genus of Saccharomyces and at least one further yeast organism selected from the genus of Saccharo- myces.
• the invention also pertains to methods for preparing the above compositions as well as to methods for their use, including the use of the compositions as starter cultures for the fermentation of a carbohydrate source during e.g. wine making.
• a method for fermenting at least one fermentable carbohydrate source in an aqueous composition comprising the step of contacting the aqueous composition with the composition according to the invention under conditions allowing said fermentation to occur.
• a method for producing an edible or drinkable product comprising the step of adding the composition according to the invention to a precursor composition of said product, fermenting said precursor composition and producing said edible or drinkable product.
• a method for flavouring an edible or drinkable product comprising the step of adding the composition according to the invention to a precursor composition of said product, fermenting said precursor composition and flavouring said edible or drinkable product.
• a method for preparing a dried composition according to the invention comprising the steps of i) providing at least one first yeast organism, and ii) drying said at least one first yeast organism to produce a dried composition thereof.
• a method for preparing a dried composition according to the invention comprising the steps of i) providing at least one first yeast organism selected from the genus of Torulaspora or the genus of Kluyveromyces, and ii) drying said composition.
• a method for preparing a dried composition according to the invention comprising the steps of i) providing a composition comprising at least one first yeast organism selected from the genus of Torulaspora and at least one second yeast species selected from the genus of Kluyveromyces, and ii) drying said composition.
• a method for preparing a dried composition according to the invention comprising the steps of i) providing a composition comprising at least one first yeast organism selected from the genus of Torulaspora or the genus of Kluyveromyces, and at least one further yeast organism selected from the genus of Saccharomyces, and ii) drying said composition.
• a method for preparing a dried composition according to the invention comprising the steps of i) providing at least one first yeast organism selected from the genus of Torulaspora and at least one second yeast organism selected from the genus of Kluyveromyces and at least one further yeast organism selected from the genus of Saccharomyces, and ii) drying said composition.
• composition according to the invention for the manufacture of a yeast starter culture for fermenting grape juice.
• composition according to the invention for producing an edible or drinkable product.
• composition according to the invention for flavouring an edible or drinkable product.
• At least one first yeast organism selected from the family of Saccharomycetaceae excluding the genus of Saccharomyces for the preparation of a yeast starter culture for fermenting grape juice and flavouring an edible or drinkable product re- suiting from said fermentation.
• composition comprising at least one first yeast organism selected from the family of Saccharomycetaceae excluding the genus of Saccharomyces and at least one second yeast organism selected from the family of Saccharomycetaceae ex- eluding the genus of Saccharomyces for the preparation of a yeast starter culture for fermenting grape juice and flavouring an edible or drinkable product resulting from said fermentation.
• composition comprising at least one first yeast organism selected from the family of Saccharomycetaceae excluding the genus of Saccharomyces and at least one further yeast organism selected from the genus of Saccharomyces for the preparation of a yeast starter culture for fermenting grape juice and flavouring an edible or drinkable product resulting from said fermentation.
• composition comprising at least one first yeast organism selected from the family of Saccharomycetaceae excluding the genus of Saccharomyces and at least one second yeast organism selected from the family of Saccharomycetaceae excluding the genus of Saccharomyces and at least one further yeast organism selected from the genus of Saccharomyces for the preparation of a yeast starter cul- ture for fermenting grape juice and flavouring an edible or drinkable product resulting from said fermentation.
• starter cultures are a yeast culture, which has been prepared for initiation of a fermentation and/or a conversion of edible or drinkable products.
• Yeast organism Any genus belonging to the family of Saccharomycetaceae. Examples include, but are not limited to: Saccharomyces, Kluyveromyces, Torulaspora, Arxiozyma, Citeromyces, Debaryomyces, Dekkera, Holleya, Issatchenkia, Kazach- stania, Kodameae, Lodderomyces, Pachysolen, Pichia, Saturnispora, Starmera, Tetrapisispora, Williopsis, Zygosaccharomyces, Brettanomyces, Kloeckera, Candida and Hanseniaspora.
• Saccharomyces Genus belonging to the family of Saccharomycetaceae.
• the genus of Saccharomyces can be characterised by e.g. rapid growing colines which are flat, smooth, glistening or dull, and cream to tannish cream in color. Pseudohyphae may be present; and when present, pseudohyphae are rudimentary. Hyphae are absent.
• Blastoconidia are 1 -celled, globose, and ellipsoid to elongate. Asci contain 1 to 4 ascospores, and do not rupture at maturity. Ascospores often are globose. Nitrate is not utilized.
• yeasts a taxonomic study. 4th ed., Elsevier, New York.
• Saccharomyces species A much preferred species is Saccharomyces cerevisiae, optionally in isolated form. Additionally preferred species includes, but is not limited to: Saccharomyces barnettii, Saccharomyces bayanus, Saccharomyces castellii, Saccharomyces dairenensis, Saccharomyces exiguus, Saccharomyces kluyveri, Saccharomyces paradoxus, Saccharomyces pastorianus, Saccharomyces rosinii, Saccharomyces servazzii, Saccharomyces spencerorum, Saccharomyces trans- vaalensis, and Saccharomyces unisporus. Saccharomyces cerevisiae deposited with the CBS under accession number CBS 1171 is claimed in connection with the present invention.
• Saccharomyces cerevisiae synonyms The following species are listed with the CBS database as synonyms of Saccharomyces cerevisiae: Candida robusta; Cryptococ- cus fermentans; Hormiscium cerevisiae; Mycotorula robusta; Saccharomyces aceti; Saccharomyces acidosaccharophillii; Saccharomyces anamensis; Saccharomyces annulatus; Saccharomyces batatae; Saccharomyces beticus; Saccharomyces bou- lardii; Saccharomyces brasiliensis; Saccharomyces busae asiaticae; Saccharomy- ces capensis; Saccharomyces carbajali; Saccharomyces carlsbergensis; Saccharomyces carlsbergensis var.
• Saccharomyces carlsbergensis var. manchuricus Saccharomyces cerevisiae Meyen ex E.G. Hansen var. cerevisiae
• Saccharomyces cerevisiae subsp. vetrozensis Saccharomyces cerevisiae var. cratericus
• Saccharomyces cerevisiae var. ellipsoideus Saccharomyces cerevisiae var. fruc- tuum
• Saccharomyces cerevisiae var. onychophilus Saccharomyces cerevisiae var. pelliculosus
• Saccharomyces cerevisiae var. pulmonalis Saccharomyces cerevisiae var. turbidans
• Saccharomyces cerevisiae agavica sylvestre Saccharomyces chevalieri var. chevalieri
• Saccharomyces chodati Saccharomyces chodati
• Saccharomyces chodati
• Saccharomyces elongatus Saccharomyces eryobotryae
• Saccharomyces formosensis Saccharomyces fructuum
• Saccharomyces gaditensis Saccharomyces hienipiensis
• Saccharomyces hispalensis Saccharomyces hispanica
• Saccharomyces hutensis Saccha- romyces ilicis
• Saccharomyces italicus var. italicus Saccharomyces italicus var. melibiosi
• Saccharomyces lindneri Saccharomyces logos
• Saccharomyces mangini var. mangini Saccharomyces mangini var. miso; Saccharomyces marchalianus; Saccharomyces melibiosi; Saccharomyces muentzii; Saccharomyces multisporus; Saccharomyces norbensis; Saccharomyces nutensis; Saccharomyces Odessa; Saccharomyces oleaceus; Saccharomyces olea- ginosus; Saccharomyces onubensis; Saccharomyces oviformis var. bisporus; Saccharomyces oviformis var. cheriensis; Saccharomyces oviformis var.
• Saccharomyces oviformis Saccharomyces oxidans; Saccharomyces pastorianus subsp. arbignensis; Saccharomyces peka; Saccharomyces prostoserdovii; Saccharomyces pulmonalis; Saccha- romyces robustus; Saccharomyces shaoshing; Saccharomyces steineri; Saccharomyces thermantitonum; Saccharomyces tokyo; Saccharomyces turbidans; Saccharomyces valesiacus; Saccharomyces veronae var.
• Saccharomyces cerevisiae As used herein. The list is non-limiting and does not exclude that further strains shall be considered synonyms of Saccharomyces cerevisiae.
• Non-Saccharomyces As used herein any of the geni belonging to the family of Sac- charomycetaceae excluding the genus of Saccharomyces. Preferred examples include, but is not limited to: Kluyveromyces, Torulaspora, Arxiozyma, Citeromyces, Debaryomyces, Dekkera, Holleya, Issatchenkia, Kazachstania, Kodameae, Lod- deromyces, Pachysolen, Pichia, Saturnispora, Starmera, Tetrapisispora, Williopsis, and Zygosaccharomyces.
• Torulaspora species A much preferred species is Torulaspora delbrueckii, optionally in isolated form. Additionally preferred species include, but is not limited to: T. alkoholi var alkoholi, T. alkoholi var. azymatica, T. amurcae, T. bailii, T. benedictae, T. bispora, T. californica, T. carsonii, T. castellii, T. cidri, T. coudertii, T. etchellsii, T. eupagyca, T. exigua, T. fermentati, T. florentina, T. formicaria, T. fransiscae, T.
• T. delbrueckii CBS 3085 is preferred.
• the genus of Torulaspora including the species of Torulaspora delbrueckii, can be characterised by e.g. rapid growing colonies which are round to oval, smooth, and white to cream in color. No hyphae are absent. Asexual reproduction is by budding. Asci contain 1 to 4 ascospores. Ascospores often are often oval and rough. Conjugation occurs between cells and between a cell and its bud.
• Torulaspora delbrueckii synonyms The following species are listed with the CBS database as synonyms of Torulaspora delbrueckii: Candida colliculosa; Cryptococ- cus colliculosus; Debaryomyces dekkerae; Debaryomyces delbrueckii; Debaryomy- ces nilssonii; Debaryomyces rosei; Eutorula colliculosa; Saccharomyces chevalieri var. torulosus; Saccharomyces delbrueckii var. delbrueckii; Saccharomyces delbrueckii var.
• Saccharomyces fermentati Saccharomyces florenzanoi
• Saccharomyces inconspicuus Saccharomyces microellipsoides var. osmophilus
• Saccharomyces nilssonii var. nilssonii
• Saccharomyces rosei Saccharomyces sai- toanus
• Saccharomyces torulosus Saccharomyces uvarum var.
• Torulopsis taboadae Zygosaccharomyces delbrueckii; Zygosaccharomyces fermentati; Zygosaccharomyces globiformis; Zygosaccharomyces globiformis f. coronata; Zygosaccharomyces globiformis f. typica; Zygosaccharomyces mongolicus; Zymodebaryomyces dek- kerae; Zymodebaryomyces delbrueckii; and Zymodebaryomyces rosei. Accordingly, the above species are also comprised by the term Torulaspora delbrueckii as used herein. The list is non-limiting and does not exclude that further strains shall be considered synonyms of Torulaspora delbrueckii.
• Kluyveromyces species A much preferred species is Kluyveromyces thermotoler- ans, optionally in isolated form. Additionally preferred species include, but is not limited to: K. subgenus Flabospora, K. subgenus Globospora, K. aestuarii, K. afri- canus, K. bacillisporus, K. blattae, K. bulgaricus, K. cellobiovorus, K. delphensis, K. dobzhanskii, K. drosophilarum, K. fragilis, K. lactis var, drosophilarum, K. lactis var. lactis, K. lodderae, K. marxianus var.
• Kluyveromyces thermotolerans CBS 2803 also available under ATCC 24177; DBVPG 6166;IFO 1674;NCYC 701 ;NRRL Y-2232;UCD 55-41 ;VKM Y-533
• CBS 7220 CBS 6925, CBS 6433, CBS 4836, CBS 4835, CBS 4834, CBS 4833, CBS 4832.
• Kluyveromyces themotolerans CBS 7220 is preferred.
• the ge- nus of Kluyveromyces, including the species of Kluyveromyces thermotolerans, can be characterised by e.g.
• yeasts a taxonomic study. 4th ed., Elsevier, New York.
• Kluyveromyces thermotolerans synonyms The following species are listed with the CBS database as synonyms of Kluyveromyces thermotolerans: Kluyveromyces Van der Walt, Kluyveromyces veronae; Kluyveromyces subgenus Fabospora; Candida dattila; Cryptococcus dattilus; Mycotorula dattila; Saccharomyces drosophilae; Sac- charomyces thermotolerans; Saccharomyces veronae var. veronae; Zygosaccharomyces drosophilae; Zygosaccharomyces thermotolerans; Torula dattila; Torulopsis dattila var.
• Kluyveromyces thermotolerans are also comprised by the term Kluyveromyces thermotolerans as used herein.
• the list is non-limiting and does not exclude that further strains shall be considered synonyms of Kluyveromyces thermotolerans.
• Yeast starter culture Culture comprising at least one yeast species in liquid culture, liquid pressed culture, frozen form or dried form, including freeze dried form and spray/fluid bed dried form.
• the culture can be packed in vacuum, or under a protected atmosphere such as e.g. nitrogen and the like.
• the culture is capable of initiating an industrial fermentation process under practical conditions, optionally after being cultivated in a separate starter medium for obtaining a high density culture.
• the culture can be added to a fermentable carbohydrate source such as e.g. grape juice once or more times during a fermentation process, and it can be added initially and/or later in the fermentation process.
• Yeast starter culture comprising at least one yeast species selected from the genus of Saccharomyces and at least one yeast species se- lected from the family of Saccharomycetaceae excluding the genus of Saccharomyces.
• Taxonomic determinations The skilled artisan has at his disposal several tools for determining which family, genus and species any particular yeast organism belongs to. For example, recent developments in molecular biology and protein chemistry have provided methods such as DNA restriction fragment length polymorphisms, protein electrophoresis patterns and chromosome fingerprinting. Such techniques have been used for identifying e.g. fermentation-related microorganisms.
• PCR Polymerase chain reaction
• ribosomal genes have been used as molecular probe targets because of their high copy number.
• Non-transcribed and transcribed spacer sequences associated with ribosomal genes are usually poorly conserved and, thus, are advantageously used as target sequences for the detection of recent evolutionary divergence.
• Fungral rRNA genes are organized in units. Each unit encodes mature subunits of 18S,
• the internal transcribed spacer (ITS) region lies between the 18S and 28S rRNA genes and contains two variable non-coding spacers (referred to as ITS1 and ITS2) and the 5.8S rRNA gene (White et al., 1990; In: PCR Protocols; Eds.: Innes et al pages 315-322).
• ITS1 and ITS2 variable non-coding spacers
• NTSs non-transcribed spacer sequences
• the figures illustrate the physiological limitations of Torulaspora delbrueckii and Kluyveromyces thermotolerans when used as pure strains, in their ability to convert the grape juice into wine, and the surprising disclosure of this invention when using mixed yeast starter cultures of Saccharomyces cerevisiae and Torulaspora delbrueckii or Saccharomyces cerevisiae and Kluyveromyces thermotolerans, or any other combinations thereof.
• grape juice each one representing an ecosystem, which is subjected to a fermentation according to the invention.
• Pinot noir grape juice has been selected as one example, but not limited to, that is subjected to red wine fermentation conditions according to the invention.
• Chardonnay grape juice has been selected as another example, but not limited to, that is subjected to white wine fermentation conditions according to the invention.
• Figure 1 illustrates a pure strain alcoholic fermentation by Saccharomyces cere- visiae in a Pinot noir grape juice (pasteurised at 140°C/284°F in 8 seconds and stored in 5 litre sterile plastic containers at 5°C/39.2°F until use) as a laboratory experiment in 3 litre scale incubated at 28°C/82.4°F.
• S. cerevisiae was prepared by fluid bed drying as generally known in the art and such as detailed in Example 2 below and re-hydrated as generally known in the art and such as detailed in Exam- pie 3 below.
• Saccharomyces cerevisiae is able, as a pure culture, to convert glucose and fructose into ethanol and glycerol in only 3 days.
• the development of CO 2 and acetic acid are not shown.
• FIG. 2 illustrates a pure strain alcoholic fermentation by Torulaspora delbrueckii in a Pinot noir grape juice (pasteurised at 140°C/284°F in 8 seconds and stored in 5 litre sterile plastic containers at 5°C/39.2°F until use) in a laboratory experiment in 3 litre scale incubated at 28°C/82.4°F.
• Torulaspora delbrueckii was prepared by fluid bed drying as generally known in the art and such as detailed in Example 2 below and re-hydrated as generally known in the art and such as detailed in Example 3 below.
• FIG. 3 illustrates a pure strain alcoholic fermentation by Kluyveromyces thermotolerans in a Pinot noir grape juice (pasteurised at 140°C/284°F in 8 seconds and stored in 5 litre sterile plastic containers at 5°C/39.2°F until use) in a laboratory experiment in 3 litre scale incubated at 28°C/82.4°F.
• Kluyveromyces thermotolerans was prepared by fluid bed drying as generally known in the art and such as detailed in Example 2 below and re-hydrated as generally known in the art and such as detailed in Example 3 below.
• K. thermotolerans as a pure culture was not able to convert all the available glucose and fructose and therefore did not complete the alcoholic fermentation during the experiment.
• the cells sustained growth for 3 days, whereafter the cell number decreased significantly.
• Figure 4 illustrates a pure strain alcoholic fermentation by Saccharomyces cerevisiae in a Chardonnay grape juice (pasteurised at 140°C/284°F in 8 seconds and stored in 5 litre sterile plastic containers at 5°C/39.2°F until use) as a laboratory experiment in 1 litre scale incubated at 20°C/68°F.
• S. cerevisiae was prepared by fluid bed drying as generally known in the art and such as detailed in Example 2 below and re-hydrated as generally known in the art and such as detailed in Example 3 below.
• Saccharomyces cerevisiae is able, as a pure culture, to con- vert glucose and fructose into ethanol and glycerol in 9 days.
• the development of C0 2 and acetic acid are not shown.
• FIG. 5 illustrates a pure strain alcoholic fermentation by Torulaspora delbrueckii in Chardonnay grape juice (pasteurised at 140°C/284°F in 8 seconds and stored in 5 litre sterile plastic containers at 5°C/39.2°F until use) in a laboratory experiment in 1 litre scale incubated at 20°C/68°F.
• Torulaspora delbrueckii was prepared by fluid bed drying as generally known in the art and such as detailed in Example 2 below and re-hydrated as generally known in the art and such as detailed in Example 3 below.
• T. delbrueckii as a pure culture did not complete the alcoholic fermentation during the experiment. The cells grew exponentially for 2 days, before entering the stationary growth phase.
• Figure 6 illustrates a pure strain alcoholic fermentation by Kluyveromyces thermotolerans in a Chardonnay juice (pasteurised at 140°C/284°F in 8 seconds and stored in 5 litre sterile plastic containers at 5°C/39.2°F until use) in a laboratory experiment in 1 litre scale incubated at 20°C/68°F.
• Kluyveromyces thermotolerans was prepared by fluid bed drying as generally known in the art and such as detailed in Ex- ample 2 below and rehydrated as generally known in the art and such as detailed in
• K thermotolerans as a pure culture did not complete the alcoholic fermentation during the experiment.
• the cells grew exponentially for 2 days, before entering the sta- tionary growth phase. No death phase appeared in this experiment.
• Saccharomyces cerevisiae was the only strain that was able to convert Pinot noir grape juice and Chardonnay grape juice into wine. Additionally, the experiments show that Torulaspora delbrueckii and Kluyveromyces thermotolerans are unable to convert the available glucose and fructose in either grape juices, thereby showing an incomplete fermentation which is not desired by wine producer.
• Torulaspora delbrueckii and Kluyvero- myces thermotolerans as single strain yeast starter cultures is not desirable in neither white wine nor red wine fermentations, due to the risk of stuck alcoholic fermentation and/or development of off-flavours and off-aromas from the indigeneous yeast and bacteria flora.
• Figure 7 illustrates an alcoholic fermentation by a mixed culture of Saccharomyces cerevisia and Torulaspora delbrueckii of Pinot noir grape juice (pasteurised at 140°C/284°F in 8 seconds and stored in 5 litre sterile plastic containers at 5°C/39.2°F until use) in a laboratory experiment in 3 litre scale incubated at 28°C/82.4°F.
• S.cerevisiae and Torulaspora delbrueckii were prepared by fluid bed drying as generally known in the art and such as detailed in Example 2 below and rehydrated as generally known in the art and such as detailed in Example 3 below.
• the 1:1 mixture of S. cerevisiae and T. delbrueckii completed the alcoholic fermentation in 3 days.
• the cell number of S. cerevisiae and T. delbrueckii, respectively, increased rapidly until day 2, where S. cerevisiae entered stationary growth phase until day 3, where the cells entered the death phase.
• the cells of T. delbrueckii were strongly affected by the growth of S. cerevisiae, and hardly grew before entering stationary growth phase, and eventually the death phase.
• Figure 8 illustrates an alcoholic fermentation by a mixed culture of Saccharomyces cerevisiae and Kluyveromyces thermotolerans of Pinot noir grape juice (pasteurised at 140°C/284°F in 8 seconds and stored in 5 litre sterile plastic containers at 5"C/39.2°F until use) in a laboratory experiment in 3 litre scale incubated at
• Saccharomyces cerevisiae and Kluyveromyces thermotolerans were prepared by fluid bed drying as generally known in the art and such as detailed in Example 2 below and rehydrated as generally known in the art and such as detailed in Example 3 below.
• Saccharomyces cerevisiae The 1 :1 mixture of Saccharomyces cerevisiae and Kluyveromyces thermotolerans completed the alcoholic fermentation in 3 days.
• the cell number of Saccharomyces cerevisiae was affected by the presence of K. thermotolerans, and grew slower before reaching stationary phase at day 2, and entered the death phase shortly there- after.
• Figure 9 illustrates an alcoholic fermentation by a mixed culture of Saccharomyces cerevisiae and Torulaspora delbrueckii of Chardonnay grape juice (pasteurised at
• Saccharomyces cerevisiae and Torulaspora delbrueckii were prepared by spray/fluid bed drying as generally known in the art and such as detailed in Ex- ample 2 below and rehydrated as generally known in the art and such as detailed in Example 3 below.
• the 1 :1 mixture of Saccharomyces cerevisiae and T. delbrueckii completed the al- coholic fermentation in 9 days.
• the cell number of Saccharomyces cerevisiae grew exponentially until day 3, before entering the stationary growth phase.
• the cells of T. delbrueckii grew exponentially until day 2, before rapidly entering the death phase.
• Figure 10 illustrates an alcoholic fermentation by a mixed culture of Saccharomyces cerevisiae and Kluyveromyces thermotolerans of Chardonnay grape juice (pasteurised at 140°C/284°F in 8 seconds and stored in 5 litre sterile plastic containers at 5°C/39.2°F until use) in a laboratory experiment in 1 litre scale incubated at 20°C/68°F.
• Saccharomyces cerevisiae and Kluyveromyces thermotolerans were prepared by spray/fluid bed drying as generally known in the art and such as de- tailed in Example 2 below and rehydrated as generally known in the art and such as detailed in Example 3 below.
• the 1 :1 mixture of Saccharomyces cerevisiae and Kluyveromyces thermotolerans completed the alcoholic fermentation in 9 days.
• the cell number of Saccharomyces cerevisiae grew exponentially until day 4, before entering the stationary growth phase.
• the cells of Kluyveromyces thermotolerans grew exponentially until day 2, before rapidly entering the death phase.
• Saccharomyces cerevisiae in combination with either Torulaspora delbrueckii or Kluyveromyces thermotolerans as mixed starter cultures showed that Saccharomyces cerevisiae is the main driving force in the alcohol fermentation.
• Torulaspora delbrueckii and Kluyveromyces thermotolerans only grew to a limited extent before entering death phase. However, this relatively short period of active growth, is sufficient to modify the aroma and flavour compounds of the finished wine, as shown in tables 1 and 2.
• the tables illustrate, but are not limited to, the aroma and flavour compounds produced by a mixture of Saccharomyces cerevisiae and Torulaspora delbrueckii or a mixture of Saccharomyces cerevisiae and Kluyveromyces thermotolerans.
• the tables illustrate the unexpected finding that the use of mixed yeast starter cultures of Saccharomyces cerevisiae and Torulaspora delbrueckii or of Saccharomyces cerevisiae and Kluyveromyces thermotolerans or any combinations thereof gives rise to the development of enhanced aroma and flavour profiles, which are different from those resulting from a pure strain fermentation of S. cerevisiae.
• the tables show aroma and flavour differences between the use of Saccharomy- ces/Torulaspora delbrueckii and Saccharomyces / Kluyveromyces thermotolerans.
• Table 1 illustrates some of the chemical aroma components produced during alcoholic fermentation of Chardonnay grape juice (pasteurised at 140°C/284°F in 8 seconds and stored in 5 litre sterile plastic containers at 5°C/39.2°F until use) by a mixed culture of Saccharomyces cerevisiae and Torulaspora delbrueckii, not related to the grape juice nor a pure strain alcoholic fermentation by Saccharomyces cerevisiae under the same conditions. The experiment was conducted in 20 litre scale incubated at 20°C/68°F. The samples were analysed by GC-mass spectrometry (Williams, D.H. & Fleming, I., 1995) and NIF (Pollien et al., 1997)
• Table 2 illustrates some of the chemical aroma components produced during alcoholic fermentation of Chardonnay grape juice by a mixed culture of Saccharomyces cerevisiae and Kluyveromyces thermotolerans not related to the grape juice nor a pure strain alcoholic fermentation by Saccharomyces cerevisiae under the same conditions. The experiment was conducted in 20 litre scale incubated at 20°C/68°F. Table 2
• Composition comprising at least one non-Saccharomvces yeast organism
• the invention when the invention according to one aspect pertains to a composition
• a composition comprising at least one first yeast organism selected from the family of Saccharomycetaceae excluding the genus of Saccharomyces, the at least one first yeast organism is in one embodiment selected from the genus of Torulaspora.
• the Torulaspora organism is preferably selected from the group consisting of T. delbrueckii, T. alcoholi var alcoholi, T. alcoholi var. azymatica, T. amurcae, T. bailii, T. benedictae, T. bispora, T. californica, T. carsonii, T. castellii, T. cidri, T. coudertii, T. etchellsii, T. eupagyca, T. exigua, T. fermentati, T. florentina, T. formicaria, T. fransiscae, T. globosa, T. hansenii, T. inconspicua, T.
• the at least one first yeast organism is in one embodiment selected from the genus of Kluyveromyces.
• the Kluyveromyces organism is preferably selected from the group consisting of K. thermotolerans, K. subgenus Flabospora, K. subgenus Globospora, K. aestuarii, K. africanus, K. bacillisporus, K. blattae, K. bulgaricus, K. cellobiovorus, K. delphensis, K. dobzhanskii, K. drosophilarum, K. fragilis, K. lactis var, drosophilarum, K. lactis var. lactis, K. lodderae, K. marxianus var. bulgaricus, K. marxianus var. dobzhanskii, K.
• marxianus var. drosophilarum K. marxianus var. lactis, K. marxianus var. marxianus, K. marxianus var. vanudenii, K. marxianus var. wikenii, K. nonfermentans, K. osmophilus, K. penaeid, K. phaffii, K. phaeseolosporus, K. piceae, K. polysporus, K. sinensis, K. vanudenii,
• K. veronae, K. waltii, K. wickerhamii, K. wikenii, and K. yarrowii including any combination thereof.
• composition is preferably freeze dried or spray/fluid bed dried.
• Composition comprising at least one first non-Saccharomvces yeast organism and at least one second non-Saccharomvces yeast organism
• the invention according to a second aspect pertains to a composition
• a composition compris- ing at least one first yeast organism selected from the family of Saccharomycetaceae excluding the genus of Saccharomyces and at least one second yeast organism selected from the family of Saccharomycetaceae excluding the genus of Saccharomyces
• the at least one first and second yeast organism is in one embodiment selected from the genus of Torulaspora and from the genus of Kluyveromyces, re- spectively.
• the Torulaspora organism is preferably selected from the group consisting of T. delbrueckii, T. alcoholi var alcoholi, T. alkoholi var. azymatica, T. amurcae, T. bailii, T. benedictae, T. bispora, T. californica, T. carsonii, T. castellii, T. cidri, T. coudertii, T. etchellsii, T. eupagyca, T. exigua, T. fermentati, T. florentina, T. formicaria, T. fransiscae, T. globosa, T. hansenii, T. inconspicua, T.
• the Kluyveromyces organism is preferably selected from the group consisting of K.
• thermotolerans K. subgenus Flabospora, K. subgenus Globospora, K. aestuarii, K. africanus, K. bacil- lisporus, K. blattae, K. bulgaricus, K. cellobiovorus, K. delphensis, K. dobzhanskii, K. drosophilarum, K. fragilis, K. lactis var, drosophilarum, K. lactis var. lactis, K. lod- derae, K. marxianus var. bulgaricus, K. marxianus var. dobzhanskii, K. marxianus var. drosophilarum, K.
• marxianus var. lactis K. marxianus var. marxianus, K. marxianus var. vanudenii, K. marxianus var. wikenii, K. nonfermentans, K. osmophilus, K. penaeid, K. phaffii, K. phaeseolosporus, K. piceae, K. polysporus, K. sinensis, K. vanudenii, K. veronae, K. waltii, K. wickerhamii, K. wikenii, K. yarrowii, including any combination thereof.
• composition wherein the Torulaspora organism is Torulaspora delbrueckii and wherein the Kluyveromy- ces organism is Kluyveromyces thermotolerans.
• composition is preferably freeze dried or spray/fluid bed dried.
• Composition comprising one or more non-Saccharomvces yeast organisms and at least one further Saccharomyces yeast organism
• the invention according to a third aspect relates to either a composition comprising at least one first yeast organism selected from the family of Saccharomycetaceae excluding the genus of Saccharomyces and at least one further yeast organism selected from the genus of Saccharomyces, or relates to a composition comprising at least one first yeast organism selected from the family of
• Saccharomycetaceae excluding the genus of Saccharomyces and at least one second yeast organism selected from the family of Saccharomycetaceae excluding the genus of Saccharomyces and at least one further yeast organism selected from the genus of Saccharomyces
• the first and/or second (non-Saccharomyces) yeast organisms can be any of the (combinations of) organisms described herein above, whereas the at least one further yeast organism is preferably selected from the group consisting of Saccharomyces cerevisiae, Saccharomyces barnettii, Saccharomyces bayanus, Saccharomyces castellii, Saccharomyces dairenensis, Saccharomyces exiguus, Saccharomyces kluyveri, Saccharomyces paradoxus, Saccharomyces pastorianus, Saccharomyces rosinii, Saccharomyces servazzii,
• Saccharomyces spencerorum Saccharomyces transvaalensis
• Saccharomyces Saccharomyces transvaalensis Saccharomyces unisporus, including any combination thereof.
• Saccharomyces cerevisiae is preferred, optionally in combination with Torulaspora delbrueckii and Kluyveromyces thermotolerans.
• composition is preferably freeze dried or spray/fluid bed dried.
• compositions are to be used as starter cultures it is desirable to have a number of colony forming units per gram (dry weight) starter culture which is preferably more than about 10 8 CFU for the at least one further yeast organism, such as more than about 10 9 CFU, for example more than about 10 10 CFU, such as more than about 2 x 10 10 CFU.
• a number of colony forming units per gram (dry weight) starter culture which is preferably more than about 10 7 CFU for the at least one first yeast organism and the same number of CFU for the at least one second yeast organism, when present, such as more than about 10 8 CFU, for example more than about 10 9 CFU, such as more than about 2 x 10 9 CFU.
• the ratio R between i) the number (CFU) of the at least one first yeast organism cells, and ii) the number (CFU) of the at least one second yeast organism cells in the composition is preferably in the range of from more than 0.1 to preferably less than 100, such as in the range of from 0.1 to 0.3, for example in the range of from 0.2 to 0.3, such as in the range of from 0.3 to 0.5, for example in the range of from 0.4 to 0.5, such as in the range of from 0.5 to 0.7, for example in the range of from 0.6 to 0.7, such as in the range of from 0.7 to 0.9, for example in the range of from 0.8 to 0.9, such as in the range of from 0.9 to 1.0 and such as about 1.0, for example in the range of from 1.0 to 1.1 , such as in the range of from 1.1 to 1.3, for example in the range of from 1.2 to 1.3, such as in the range of
• the ratio R between i) the number (CFU) of the at least one first yeast organism cells, and ii) the number (CFU) of the at least one further yeast organism cells is preferably in the range of from more than 0.1 to preferably less than 100, such as in the range of from 0.1 to 0.3, for example in the range of from 0.2 to 0.3, such as in the range of from 0.3 to 0.5, for example in the range of from 0.4 to 0.5, such as in the range of from 0.5 to 0.8, for example in the range of from 0.6 to 0.8, such as in the range of from 0.7 to 0.9, for example in the range of from 0.8 to 0.9, such as in the range of from 0.9 to 1.0 and such as about 1.0, for example in the range of from 1.0 to 1.1 , such as in the range of from 1.1 to 1.3, for example in the range of from 1.2 to 1.3, such as in the range of from 1.3 to
• 1.5 for example in the range of from 1.4 to 1.5, such as in the range of from 1.6 to 1.8, for example in the range of from 1.7 to 1.8, such as in the range of from 1.8 to 2.0, for example in the range of from 1.9 to 2.0, such as in the range of from 2.0 to 2.4, for example in the range of from 2.2 to 2.4, such as in the range of from 2.4 to 3.0, for example in the range of from 2.8 to 3.0, such as in the range of from 3.0 to
• 4.0 for example in the range of from 3.5 to 4.0, such as in the range of from 4.0 to 5.0, for example in the range of from 4.5 to 5.0, such as in the range of from 5.0 to 7.0, for example in the range of from 6.0 to 7.0, such as in the range of from 7.0 to 9.0, for example in the range of from 8.0 to 9.0, such as in the range of from 9.0 to 12, for example in the range of from 10 to 12, such as in the range of from 12 to 16, for example in the range of from 14 to 16, such as in the range of from 16 to 20, for example in the range of from 18 to 20, such as in the range of from 20 to 30, for example in the range of from 25 to 30, such as in the range of from 30 to 50, for example in the range of from 40 to 50, and such as in the range of from 50 to 100.
• the ratio R between i) the number (CFU) of cells of the at least one first yeast organism and the at least one second yeast organism, and ii) the number (CFU) of the at least one further yeast organism cells is preferably in the range of from more than 0.1 to preferably less than 100, such as in the range of from 0.1 to 0.3, for example in the range of from 0.2 to 0.3, such as in the range of from 0.3 to 0.5, for example in the range of from 0.4 to 0.5, such as in the range of from 0.5 to 0.8, for example in the range of from 0.6 to 0.8, such as in the range of from 0.7 to 0.9, for example in the range of from 0.8 to 0.9, such as in the range of from 0.9 to 1.0 and such as about 1.0, for example in the range of from 1.0 to 1.1, such as in the range of from 1.1 to 1.3, for example in the range of from 1.2 to 1.3
• the ratios (in colony forming units, CFU) among the aforementioned species a):b):c) is in one embodiment preferably about 1:1:1, such as about 1:1:2; for example 1:1:3, such as about 1:1:4; for example 1 :1 :5; such as about 1:2:1 , for example 1 :2:2; such as about 1 :2:3, for example 1 :2:4; such as about 1 :2:5, for example 1:3:1 ; such as about 1 :3:2, for example 1 :3:3; such as about 1 :3:4, for example 1 :3:5; such as about 1:4:1, for example
• the invention also relates to a method for fermenting at least one fermentable carbohydrate source in an aqueous composition.
• This method comprises the step of contacting the aqueous composition with the composition of the invention under conditions allowing said fermentation to occur, and in this case the carbohydrate source preferably comprises at least one pentose or at least one hexose, including any monomer, dimer, oligomer or polymer thereof.
• Preferred fermentable carbohydrate sources are e.g. fructose, glucose, galactose, maltose, sucrose, trehalose, melibiose, starch, raffinose, including any combination thereof.
• other carbohydrate sources can also be used.
• the carbohydrate sources can be obtained from white grapes or red grapes, or both.
• the white grapes can be selected from the group of grapes consisting of Aligote, Chardonnay, Chenin Blanc, Columbard, Folle Blanche, Gewurztraminer, Gr ⁇ ner Veltliner, Malvasia, Marsanne, Melon de Bourgogne, Muller-Thurgau, Muscadelle, Muscat, Palomino, Pedro Xime- nez, Pinot Blanc, Pinot Gris/Pinot Grigio, Riesling, Rousanne, Sauvignon
• Blanc/Fume Blanc Scheurebe, Semillion, Sylvaner, Trebbiano, Ugni Blanc, Verdic- chio, Viognier, including any combination thereof.
• the red grapes can be selected from the group of grapes consisting of Barbera, Brunello, Cabernet Franc, Cabernet Sauvignon, Carignana, Carmenere, Cinsault,
• the invention relates to a method for producing an edible or drinkable product, said method comprising the step of adding the composition according to the invention to a precursor composition of said product, fermenting said precursor composition and producing said edible or drinkable product, the product is in one embodi- ment an alcoholic beverage including wine, such as red wine, rose wine, blush wine, white wine, sparkling wine, and fruit wine. Further examples of alcoholic beverages comprise cider and beer. Examples of edible products comprise bread. Further examples of products are milk and soy.
• the invention in another embodiment, relates to a method for flavouring an edible or drinkable product, said method comprising the step of adding the composition according to the invention to a precursor composition of said product, fermenting said precursor composition and flavouring said edible or drinkable product, preferably an alcoholic beverage such as e.g. wine, for example red wine, rose wine, blush wine, white wine, sparkling wine, and fruit wine, as well as cider and beer.
• an alcoholic beverage such as e.g. wine, for example red wine, rose wine, blush wine, white wine, sparkling wine, and fruit wine, as well as cider and beer.
• the flavour can also be added to e.g. bread, milk and soy.
• the invention relates to a method for preparing a dried composition according to the invention, said method comprising the steps of i) providing at least one first yeast organism, and ii) drying said at least one first yeast organism to produce a dried composition, preferably a freeze dried composition or a spray/fluid bed dried composition.
• the method in one preferred embodiment comprises the steps of i) providing at least one first yeast organism selected from the genus of Torulaspora or the genus of Kluyveromyces, and ii) drying said composition, preferably by freeze drying or by spray/fluid bed drying.
• the first yeast organism is preferably Torulaspora delbrueckii or Kluyveromyces thermotolerans.
• the method in another preferred embodiment comprises the steps of i) providing a composition comprising at least one first yeast organism selected from the genus of Torulaspora and at least one second yeast species selected from the genus of Kluyveromyces, and ii) drying said composition, preferably by freeze drying or by spray/fluid bed drying.
• the first yeast organism is preferably Torulaspora delbrueckii and the second yeast organism is preferably Kluyveromyces thermotolerans.
• the method in yet another preferred embodiment comprises the steps of i) providing a composition comprising at least one first yeast organism selected from the genus of Torulaspora or the genus of Kluyveromyces, and at least one further yeast organism selected from the genus of Saccharomyces, and ii) drying said composition, preferably by freeze drying or by spray/fluid bed drying.
• the first yeast organism is preferably Torulaspora delbrueckii or Kluyveromyces thermotolerans
• the at least one further yeast organism is preferably Saccharomyces cerevisiae.
• the method in an even further preferred embodiment comprises the steps of i) providing at least one first yeast organism selected from the genus of Torulaspora and at least one second yeast organism selected from the genus of Kluyveromyces and at least one further yeast organism selected from the genus of Saccharomyces, and ii) drying said composition, preferably by freeze drying or by spray/fluid bed drying.
• the first yeast organism is preferably Torulaspora delbrueckii
• the second yeast organism is preferably Kluyveromyces thermotolerans
• the further yeast organism is preferably Saccharomyces cerevisiae.
• composition according to the invention for the manufacture of a yeast starter culture for fermenting grape juice including grape juice obtained from white grapes such as e.g. Aligote, Chardonnay, Chenin Blanc, Columbard, Folle Blanche, Gewurztraminer, Gr ⁇ ner Veltliner, Malva- sia, Marsanne, Melon de Bourgogne, Muller-Thurgau, Muscadelle, Muscat, Palo- mino, Pedro Ximenez, Pinot Blanc, Pinot Gris/Pinot Grigio, Riesling, Rousanne,
• white grapes such as e.g. Aligote, Chardonnay, Chenin Blanc, Columbard, Folle Blanche, Gewurztraminer, Gr ⁇ ner Veltliner, Malva- sia, Marsanne, Melon de Bourgogne, Muller-Thurgau, Muscadelle, Muscat, Palo- mino, Pedro Ximenez, Pinot Blanc, Pinot Gris/Pinot Grigio, Riesling, Rousanne,
• the invention also relates to the use of a composition according to the invention for producing an edible or drinkable product.
• a composition according to the invention for flavouring an edible or drinkable product.
• the products include alcoholic beverages, including wine such as red wine, rose wine, blush wine, white wine, sparkling wine, fruit wine, as well as cider and beer. Additional products capable of being produced by the invention are bread, milk and soy.
• At least one first yeast organism selected from the family of Saccharomycetaceae excluding the genus of Saccharomyces for the preparation of a yeast starter culture for fermenting grape juice and flavouring an edible or drinkable product resulting from said fermentation.
• the starter culture is preferably dried such as freeze dried or /fluid bed dried.
• the first yeast organism is preferably selected from the genus of Torulaspora, such as Torulaspora delbrueckii.
• the first species can also be selected from the genus of Kluyveromyces, including Kluyveromyces thermotolerans.
• the starter culture is preferably dried by freeze drying or by spray/fluid bed drying when the present invention is used to provide a composition comprising at least one first yeast organism selected from the family of Saccharomycetaceae excluding the genus of Saccharomyces and at least one second yeast organism selected from the family of Saccharomycetaceae excluding the genus of Saccharomyces for the preparation of a yeast starter culture for fermenting grape juice and flavouring an edible or drinkable product resulting from said fermentation.
• the first yeast organism is preferably selected from the genus of Torulaspora, including Torulaspora del- brueckii, whereas the second yeast organism is preferably selected from the genus of Kluyveromyces, including Kluyveromyces thermotolerans.
• the starter culture is preferably dried by freeze drying or by spray/fluid bed drying when the present invention is used to provide a composition comprising at least one first yeast organism selected from the family of Saccharomycetaceae excluding the genus of Saccharomyces and at least one further yeast organism selected from the genus of Saccharomyces for the preparation of a yeast starter culture for fermenting grape juice and flavouring an edible or drinkable product resulting from said fermentation.
• the first yeast organism is preferably selected from the genus of Torulaspora, such as Torulaspora delbrueckii.
• the first species can also be selected from the genus of Kluyveromyces, including Kluyveromyces thermotolerans.
• the further yeast organism is preferably Saccharomyces cerevisiae.
• composition comprising at least one first yeast organism selected from the family of Saccharomycetaceae excluding the genus of Saccharomyces and at least one second yeast organism selected from the family of Saccharomycetaceae excluding the genus of Saccharomyces and at least one further yeast organism selected from the genus of Saccharomyces for the preparation of a yeast starter culture for fermenting grape juice and flavouring an edible or drinkable product resulting from said fermen- tation.
• the prepared starter culture is preferably dried, such as freeze dried or spray/fluid bed dried.
• the first yeast organism is preferably selected from the genus of Torulaspora, including Torulaspora delbrueckii, and the second yeast organism is pref- erably selected from the genus of Kluyveromyces, including Kluyveromyces thermotolerans.
• the further yeast organism is preferably Saccharomyces cerevisiae.
• starter cultures is to be understood as inoculation material of the appropriate yeast strain for large-scale production.
• Preparation of starter culture for wine fermentation bv means of freeze drying
• starter culture consisting of either Saccharomyces cerevisiae, or Torulaspora delbrueckii, or Kluyveromyces thermotolerans.
• YPG growth medium Preparation of YPG growth medium.
• YPG agar Preparation of YPG agar.
• the liquid agar solution is dispersed in sterile petri dishes stored at 5°C until use.
• Step 1 A pure culture ampoule containing the appropriate yeast species kept at
• -80°C/-112°F is dispersed on an YPG agar plate and incubated for 48 hours at 30°C/86°F.
• Step 2 From this YPG agar plate, a single colony of the appropriate yeast species is picked, and transferred to an YPG medium. The medium is incubated for 48 hours at 30°C/86°F under shaking (200 rpm).
• Step 3 This yeast solution is used for preparing a 1 % starter culture in semi-large scale to fit an starter culture of 1% in the final large-scale.
• the 1% starter culture is prepared by diluting in a ratio of 1 to 100 the starter culture with the media used for the large scale production.
• 1% inoculation material prepared as described above is inoculated into production tank containing YPG growth medium.
• Glucose being the carbohydrate source can be substituted by e.g. molasses or starch.
• the cells are incubated at 30°C/86°F while being stirred at 200 rpm.
• the carbohydrate source is pumped into a tank while correlating with monitoring the optical density and air is pumped into tank while cor- relating with the ethanol production, i.e. if it is possible to measure the presence of any ethanol, more oxygen is to be pumped into the tank.
• Inlets of air flow and carbohydrate are stopped after 16-18 hours. Cells are ready to be harvested after 24 hours.
• the cells are harvested and concentrated approximately 15 times by centrifugation (e.g. 4000 rpm, 10 minutes). 1 % glycerol is added as cryoprotective agent.
• the concentrated cells are poured onto sterile trays and placed in a freeze dryer under vacuum. Water is evaporated from the cells monitored by weight loss during the drying. When the weight becomes constant, the product is dry, and is distributed to packing of sterile aluminium bags in various sizes as follows.
• Freeze dried products of Saccharomyces cerevisiae and T. delbrueckii are mixed in a desired ratio as described on pp , in package sizes of e.g. 500 g, 1 kg, 5 kg, 25 kg.
• Freeze dried products of Saccharomyces cerevisiae and K thermotolerans are mixed in a desired ratio as described, in package sizes of e.g. 500 g, 1 kg, 5 kg, 25 kg.
• Freeze dried products of Saccharomyces cerevisiae, T. delbrueckii and K. thermotolerans are mixed in a desired ratio as described, in package sizes of e.g. 500 g, 1 kg, 5 kg, 25 kg.
• the following example illustrates the production and spray/fluid bed drying of Saccharomyces cerevisiae, Torulaspora delbrueckii, and Kluyveromyces thermotolerans.
• Preparation of YPG growth medium A mixture of 10 g/L D-Glucose, 5 g/L yeast extract, and 10 g/L peptone (YPG) adjusted to pH 5.5 adding 1 N HCl before auto- clavation at 121 °C/249.8°F for 20 minutes.
• YPG agar Preparation of YPG agar.
• the liquid agar solution is dispersed in sterile petri dishes stored at 5°C until use.
• Step 1 A pure culture ampoule containing the appropriate yeast species kept at -80°C/-112°F is dispersed on an YPG agar plate an incubated for 48 hours at
• Step 2 From this YPG agar plate, a single colony of the appropriate yeast species is picked, and transferred to an YPG medium. The medium is incubated for 48 hours at 30°C/86°F under shaking (200 rpm). Step 3.
• This yeast solution is used for preparing a 1% starter culture in semi-large scale to fit an starter culture of 1% in the final large-scale.
• the 1% starter culture is prepared by diluting in a ratio of 1 to 100 the starter culture with the media used for the large scale production.
• 1% inoculation material prepared as described above is inoculated into production tank containing YPG growth medium.
• Glucose being the carbohydrate source can be substituted by e.g. molasses or starch.
• the cells are incubated at 30°C/86°F while being stirred at 200 rpm.
• the carbohydrate source is pumped into a tank while correlating with monitoring the optical density and air is pumped into tank while correlating with the ethanol production, i.e. if ethanol is measured more oxygen is pumped into the tank. Inlets of air flow and carbohydrate are stopped after 16-18 hours. Cells are ready to be harvested after 24 hours.
• the cells are harvested and concentrated approximately 15 times by centrifugation (e.g. 4000 rpm, 10 minutes). 1% glycerol is added as cryoprotective agent.
• the concentrated cells is further up-concentrated in a fluid bed and thereafter pressed through an extruder or by spray drying directly into the dryer.
• the resulting yeast paste is then fed into a drying tower where the cells are dried.
• the product is dry, it is distributed to packings of sterile aluminium bags in various sizes as follows.
• Spray/fluid bed dried products of Saccharomyces cerevisiae and T. delbrueckii are mixed in a desired ratio as described on pp, in package sizes of e.g. 500 g, 1 kg, 5 kg, 25 kg.
• Spray/fluid bed products of Saccharomyces cerevisiae and K. thermotolerans are mixed in a desired ratio as described on pp, in package sizes of e.g. 500 g, 1 kg, 5 kg, 25 kg.
• Spray/fluid bed products of Saccharomyces cerevisiae, T. delbrueckii and K ther- motolerans are mixed in a desired ratio as described on pp, in package sizes of e.g. 500 g, 1 kg, 5 kg, 25 kg.
• a sealed pouch is opened containing the dried mixed yeast starter culture of Saccharomyces cerevisiae and T. delbrueckii.
• a dosage of 10-20 g/hl grape juice to be fermented is transferred into a mixture of 1/3 of grape juice and 2/3 of tap water preheated to 35°C/95°F at mixed. Leave without stirring for 20 minutes, then homogenize properly, and add into the grape juice during pumping.
• the carbohydrates in the grape juice are then fermented into end-fermentation product by Saccharomyces cerevisiae and T. delbrueckii. T. delbrueckii will only be active in the first part of the alcoholic fermentation until the oxygen is depleted. Saccharomyces cerevisiae will be active through the alcoholic fermentation and will be responsible for the completion of the alcoholic fermentation.
• the recommended dosage is corresponding to a final concentration of yeast cells of 1-2 x 10 6 /ml.
• the sealed pouch is opened containing the dried mixed yeast starter culture of Saccharomyces cerevisiae and K. thermotolerans.
• a dosage of 10-20 g/hl grape juice to be fermented is transferred into a mixture of 1/3 of grape juice and 2/3 of tap water preheated to 35°C/95°F at mixed. Leave without stirring for 20 minutes, then homogenize properly, and add into the grape juice during pumping. Fermentation
• the carbohydrates in the grape juice are then fermented into end-fermentation product by Saccharomyces cerevisiae and K. thermotolerans.
• K. thermotolerans will only be active in the first part of the alcoholic fermentation until the oxygen is depleted. Saccharomyces cerevisiae will be active through the alcoholic fermentation and will be responsible for the completion of the alcoholic fermentation.
• the recommended dosage is corresponding to a final concentration of yeast cells of 1-2 x 10 6 /ml.
• the sealed pouch is opened containing the dried mixed yeast starter culture of Saccharomyces cerevisiae and Candida.
• a dosage of 10-20 g/hl grape juice to be fermented is transferred into a mixture of 1/3 of grape juice and 2/3 of tap water preheated to 35°C/95°F at mixed. Leave without stirring for 20 minutes, then homogenize properly, and add into the grape juice during pumping.
• the carbohydrates in the grape juice are then fermented into end-fermentation product by Saccharomyces cerevisiae and Candida.
• Candida will only be active in the first part of the alcoholic fermentation until the oxygen is depleted.
• Saccharomyces cerevisiae will be active through the alcoholic fermentation and will be responsible for the completion of the alcoholic fermentation.
• the recommended dosage is corresponding to a final concentration of yeast cells of 1-2 x 10 6 /ml.
• the sealed pouch is opened containing the dried mixed yeast starter culture of Saccharomyces cerevisiae and Dekkera/Brettannomyces.
• a dosage of 10-20 g/hl grape juice to be fermented is transferred into a mixture of 1/3 of grape juice and 2/3 of tap water preheated to 35°C/95°F at mixed. Leave without stirring for 20 minutes, then homogenize properly, and add into the grape juice during pumping.
• the carbohydrates in the grape juice are then fermented into end-fermentation product by Saccharomyces cerevisiae and Dekkera/Brettannomyces.
• Dekkera/Brettannomyces will only be active in the first part of the alcoholic fermentation until the oxygen is depleted.
• Saccharomyces cerevisiae will be active through the alcoholic fermentation and will be responsible for the completion of the alcoholic fermentation.
• the recommended dosage is corresponding to a final concentration of yeast cells of 1-2 x 10 6 /ml.
• the sealed pouch is opened containing the dried mixed yeast starter culture of Sac- charomyces cerevisiae and Pichia.
• a dosage of 10-20 g/hl grape juice to be fermented is transferred into a mixture of 1/3 of grape juice and 2/3 of tap water preheated to 35°C/95°F at mixed. Leave without stirring for 20 minutes, then homogenize properly, and add into the grape juice during pumping.
• Saccharomyces cerevisiae and Pichia will only be active in the first part of the alcoholic fermentation until the oxygen is depleted. Saccharomyces cerevisiae will be active through the alcoholic fermentation and will be responsible for the completion of the alcoholic fermentation.
• the recommended dosage is corresponding to a final concentration of yeast cells of 1-2 x 10 6 /ml.
• the sealed pouch is opened containing the dried mixed yeast starter culture of Saccharomyces cerevisiae and Metschnikowia.
• a dosage of 10-20 g/hl grape juice to be fermented is transferred into a mixture of 1/3 of grape juice and 2/3 of tap water preheated to 35°C/95°F at mixed. Leave without stirring for 20 minutes, then homogenize properly, and add into the grape juice during pumping.
• the carbohydrates in the grape juice are then fermented into end-fermentation product by Saccharomyces cerevisiae and Metschnikowia. Metschnikowia will only be active in the first part of the alcoholic fermentation until the oxygen is depleted. Saccharomyces cerevisiae will be active through the alcoholic fermentation and will be responsible for the completion of the alcoholic fermentation.
• the sealed pouch is opened containing the dried mixed yeast starter culture of Sac- charomyces cerevisiae, T. delbrueckii and K. thermotolerans.
• a dosage of 10-20 g/hl grape juice to be fermented is transferred into a mixture of 1/3 of grape juice and 2/3 of tap water preheated to 35°C/95°F at mixed. Leave without stirring for 20 minutes, then homogenize properly, and add into the grape juice during pumping.
• the carbohydrates in the grape juice are then fermented into end-fermentation product by Saccharomyces cerevisiae, T. delbrueckii and K thermotolerans.
• T. delbrueckii and K. thermotolerans will only be active in the first part of the alcoholic fermentation until the oxygen is depleted.
• Saccharomyces cerevisiae will be active through the alcoholic fermentation and will be responsible for the completion of the alcoholic fermentation.
• the recommended dosage is corresponding to a final concentration of yeast cells of 1-2 x 10 6 /ml.
• the sealed pouch is opened containing the dried mixed yeast starter culture of Saccharomyces cerevisiae, K thermotolerans and Candida.
• a dosage of 10-20 g/hl grape juice to be fermented is transferred into a mixture of 1/3 of grape juice and 2/3 of tap water preheated to 35°C/95°F at mixed. Leave without stirring for 20 minutes, then homogenize properly, and add into the grape juice during pumping.
• Saccharomyces cerevisiae The carbohydrates in the grape juice are then fermented into end-fermentation product by Saccharomyces cerevisiae, K thermotolerans and Candida.
• K. thermotolerans and Candida will only be active in the first part of the alcoholic fermentation until the oxygen is depleted. Saccharomyces cerevisiae will be active through the alcoholic fermentation and will be responsible for the completion of the alcoholic fermentation.
• the recommended dosage is corresponding to a final concentration of yeast cells of 1-2 x 10 6 /ml.
• the sealed pouch is opened containing the dried mixed yeast starter culture of Saccharomyces cerevisiae, K. thermotolerans and Dekkera/Brettannomyces.
• a dosage of 10-20 g/hl grape juice to be fermented is transferred into a mixture of 1/3 of grape juice and 2/3 of tap water preheated to 35°C/95°F at mixed. Leave without stirring for 20 minutes, then homogenize properly, and add into the grape juice during pumping.
• the carbohydrates in the grape juice are then fermented into end-fermentation product by Saccharomyces cerevisiae, K. thermotolerans and Dekkera/Brettannomyces.
• K. thermotolerans and Dekkera/Brettannomyces will only be active in the first part of the alcoholic fermentation until the oxygen is depleted.
• Saccharomyces cerevisiae will be active through the alcoholic fermentation and will be responsible for the completion of the alcoholic fermentation.
• the sealed pouch is opened containing the dried mixed yeast starter culture of Sac- charomyces cerevisiae, K. thermotolerans and Pichia.
• a dosage of 10-20 g/hl grape juice to be fermented is transferred into a mixture of 1/3 of grape juice and 2/3 of tap water preheated to 35°C/95°F at mixed. Leave without stirring for 20 minutes, then homogenize properly, and add into the grape juice during pumping.
• the carbohydrates in the grape juice are then fermented into end-fermentation product by Saccharomyces cerevisiae, K. thermotolerans and Pichia.
• K. thermotolerans and Pichia will only be active in the first part of the alcoholic fermentation until the oxygen is depleted.
• Saccharomyces cerevisiae will be active through the alcoholic fermentation and will be responsible for the completion of the alcoholic fermentation.
• the recommended dosage is corresponding to a final concentration of yeast cells of 1-2 x 10 6 /ml.
• the sealed pouch is opened containing the dried mixed yeast starter culture of Saccharomyces cerevisiae, K. thermotolerans and Metschnikowia.
• a dosage of 10-20 g/hl grape juice to be fermented is transferred into a mixture of 1/3 of grape juice and 2/3 of tap water preheated to 35°C/95°F at mixed. Leave without stirring for 20 minutes, then homogenize properly, and add into the grape juice during pumping.
• the recommended dosage is corresponding to a final concentration of yeast cells of 1-2 x 10 6 /ml.
• the sealed pouch is opened containing the dried mixed yeast starter culture of Saccharomyces cerevisiae, Candida and Dekkera/Brettannomyces.
• a dosage of 10-20 g/hl grape juice to be fermented is transferred into a mixture of 1/3 of grape juice and 2/3 of tap water preheated to 35°C/95°F at mixed. Leave without stirring for 20 minutes, then homogenize properly, and add into the grape juice during pumping.
• the carbohydrates in the grape juice are then fermented into end-fermentation product by Saccharomyces cerevisiae, Candida and Dekkera/Brettannomyces.
• Candida and Dekkera/Brettannomyces will only be active in the first part of the alcoholic fermentation until the oxygen is depleted.
• Saccharomyces cerevisiae will be active through the alcoholic fermentation and will be responsible for the completion of the alcoholic fermentation.
• the sealed pouch is opened containing the dried mixed yeast starter culture of Sac- charomyces cerevisiae, Candida and Pichia.
• a dosage of 10-20 g/hl grape juice to be fermented is transferred into a mixture of 1/3 of grape juice and 2/3 of tap water preheated to 35°C/95°F at mixed. Leave without stirring for 20 minutes, then homogenize properly, and add into the grape juice during pumping.
• the carbohydrates in the grape juice are then fermented into end-fermentation product by Saccharomyces cerevisiae, Candida and Pichia.
• Candida and Pichia will only be active in the first part of the alcoholic fermentation until the oxygen is depleted.
• Saccharomyces cerevisiae will be active through the alcoholic fermentation and will be responsible for the completion of the alcoholic fermentation.
• the recommended dosage is corresponding to a final concentration of yeast cells of 1-2 x 10 6 /ml.
• the sealed pouch is opened containing the dried mixed yeast starter culture of Saccharomyces cerevisiae, Candida and Metschnikowia.
• a dosage of 10-20 g/hl grape juice to be fermented is transferred into a mixture of 1/3 of grape juice and 2/3 of tap water preheated to 35°C/95°F at mixed. Leave without stirring for 20 minutes, then homogenize properly, and add into the grape juice during pumping.
• Saccharomyces cerevisiae The carbohydrates in the grape juice are then fermented into end-fermentation product by Saccharomyces cerevisiae, Candida and Metschnikowia.
• Candida and Metschnikowia will only be active in the first part of the alcoholic fermentation until the oxygen is depleted.
• Saccharomyces cerevisiae will be active through the alcoholic fermentation and will be responsible for the completion of the alcoholic fermentation.
• the recommended dosage is corresponding to a final concentration of yeast cells of 1-2 x 10 6 /ml.
• the sealed pouch is opened containing the dried mixed yeast starter culture of Sac- charomyces cerevisiae, Dekkera/Brettannomyces and Pichia.
• a dosage of 10-20 g/hl grape juice to be fermented is transferred into a mixture of 1/3 of grape juice and 2/3 of tap water preheated to 35°C/95°F at mixed. Leave without stirring for 20 minutes, then homogenize properly, and add into the grape juice during pumping.
• Saccharomyces cerevisiae The carbohydrates in the grape juice are then fermented into end-fermentation product by Saccharomyces cerevisiae, Dekkera/Brettannomyces and Pichia.
• Dekkera/Brettannomyces and Pichia will only be active in the first part of the alcoholic fermentation until the oxygen is depleted.
• Saccharomyces cerevisiae will be active through the alcoholic fermentation and will be responsible for the completion of the alcoholic fermentation.
• the recommended dosage is corresponding to a final concentration of yeast cells of 1-2 x 10 6 /ml.
• the sealed pouch is opened containing the dried mixed yeast starter culture of Saccharomyces cerevisiae, Dekkera/Brettannomyces and Metschnikowia.
• a dosage of 10-20 g/hl grape juice to be fermented is transferred into a mixture of 1/3 of grape juice and 2/3 of tap water preheated to 35°C/95°F at mixed. Leave without stirring for 20 minutes, then homogenize properly, and add into the grape juice during pumping.
• Saccharomyces cerevisiae The carbohydrates in the grape juice are then fermented into end-fermentation product by Saccharomyces cerevisiae, Dekkera/Brettannomyces and Metschnikowia.
• Dekkera/Brettannomyces and Metschnikowia will only be active in the first part of the alcoholic fermentation until the oxygen is depleted.
• Saccharomyces cerevisiae will be active through the alcoholic fermentation and will be responsible for the completion of the alcoholic fermentation.
• the recommended dosage is corresponding to a final concentration of yeast cells of 1-2 x 10 6 /ml.
• the sealed pouch is opened containing the dried mixed yeast starter culture of Saccharomyces cerevisiae, T. delbrueckii and Candida.
• a dosage of 10-20 g/hl grape juice to be fermented is transferred into a mixture of 1/3 of grape juice and 2/3 of tap water preheated to 35°C/95°F at mixed. Leave without stirring for 20 minutes, then homogenize properly, and add into the grape juice during pumping.
• the recommended dosage is corresponding to a final concentration of yeast cells of 1-2 x 10 6 /ml.
• the sealed pouch is opened containing the dried mixed yeast starter culture of Saccharomyces cerevisiae, T. delbrueckii and Dekkera/Brettannomyces.
• a dosage of 10-20 g/hl grape juice to be fermented is transferred into a mixture of 1/3 of grape juice and 2/3 of tap water preheated to 35°C/95°F at mixed. Leave without stirring for 20 minutes, then homogenize properly, and add into the grape juice during pumping.
• the carbohydrates in the grape juice are then fermented into end-fermentation product by Saccharomyces cerevisiae, T. delbrueckii and Dekkera/Brettannomyces.
• T. delbrueckii and Dekkera/Brettannomyces will only be active in the first part of the alcoholic fermentation until the oxygen is depleted.
• Saccharomyces cerevisiae will be active through the alcoholic fermentation and will be responsible for the completion of the alcoholic fermentation.
• the recommended dosage is corresponding to a final concentration of yeast cells of 1-2 x 10 6 /ml.
• the sealed pouch is opened containing the dried mixed yeast starter culture of Saccharomyces cerevisiae, T. delbrueckii and Pichia.
• a dosage of 10-20 g/hl grape juice to be fermented is transferred into a mixture of 1/3 of grape juice and 2/3 of tap water preheated to 35°C/95°F at mixed. Leave without stirring for 20 minutes, then homogenize properly, and add into the grape juice during pumping.
• the carbohydrates in the grape juice are then fermented into end-fermentation product by Saccharomyces cerevisiae, T. delbrueckii and Pichia.
• T. delbrueckii and Pichia will only be active in the first part of the alcoholic fermentation until the oxygen is depleted.
• Saccharomyces cerevisiae will be active through the alcoholic fer- mentation and will be responsible for the completion of the alcoholic fermentation.
• the recommended dosage is corresponding to a final concentration of yeast cells of 1-2 x 10 6 /ml.
• the sealed pouch is opened containing the dried mixed yeast starter culture of Saccharomyces cerevisiae, T. delbrueckii and Metschnikowia.
• a dosage of 10-20 g/hl grape juice to be fermented is transferred into a mixture of 1/3 of grape juice and 2/3 of tap water preheated to 35°C/95°F at mixed. Leave without stirring for 20 minutes, then homogenize properly, and add into the grape juice during pumping.
• the carbohydrates in the grape juice are then fermented into end-fermentation product by Saccharomyces cerevisiae, T. delbrueckii and Metschnikowia.
• T. del- brueckii and Metschnikowia will only be active in the first part of the alcoholic fer- mentation until the oxygen is depleted.
• Saccharomyces cerevisiae will be active through the alcoholic fermentation and will be responsible for the completion of the alcoholic fermentation.

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