Abstract:
The present invention provides a new wine yeast product in a frozen form. The product is produced in a fermenter, concentrated, cryoprotectants are added. This mixture is then frozen at −50° C. What makes this product unique is that besides the fact that it is frozen, it can be directly added to grape juice as no rehydration is required because the yeast was not dehydrated in the production process.

Description:
FIELD OF THE INVENTION 
       [0001]    The present invention relates to a method for inoculating yeast into fruit juice or fruit. This is important for the beverage industry in general, especially for the wine industry. 
       BACKGROUND 
       [0002]    For the last 50 years winemakers have become accustomed to inoculation their wine with pure cultures of yeast. Such inoculation cultures may also be termed “starter cultures”. The yeast is in a dehydrated form called active dried yeast (ADY) which has been produced in fermenters, concentrated and then dried on a drum, a process called fluid bed drying. It may herein also be termed freeze-dried yeast. 
         [0003]    As a result of the fact that the yeast is dehydrated, it therefore needs to be rehydrated, in order to be metabolically active, before application to the substrate such as grape juice as in the case of winemaking. This is a very delicate process for the yeast cells and requires a significant amount of attention and care to make sure that the yeast cell hydrates in a way that makes the cell viable. 
         [0004]    As discussed on page  124 , left column of the text book of Roger Boulton et al, 1996—during the drying process of active dried yeast production, the cellular membranes of the yeast cells loose their permeability barrier function. Therefore, to re-establish this function, it is important to re-hydrate the membranes by adding the yeast in water at 40° C. for 20 minutes. Therefore, the yeast re-hydration process typically involves a 20-30 minute rehydration in un-chlorinated water or a water/grape juice mix (2:1) between 35-38° C. then followed by the addition of grape juice of the same volume (50:50 juice/water blend) which is kept for another 20-30 minutes before adding it to wine. It is important that the grape juice should not contain any SO 2 , which could kill the yeast cells during the sensitive process of rehydration (O&#39;Kennedy, 2008). In addition, the rehydration mix must be cooled down with juice after 20 min in water, 5° C. at a time. Failure to cool down from the rehydration temperature after 30 minutes can also result in significant cell death (O&#39;Kennedy, 2008). Furthermore, care should be taken to use uncontaminated grape juice for the rehydration protocol as rehydration with contaminated grape juice will result in contamination of all wine fermentation inoculated using the rehydration mixture. Different manufacturers propose variations on this protocol but the critical step is that dehydrated cells need to be exposed to water or a water/juice mixture at specific temperatures, conditions and for specific times in order to hydrate properly, thereby avoiding cell death and consequent in-activity. It is not recommended to add the active dried yeast directly to wine as the high sugar concentration, SO 2  and other compounds in grape juice do not allow for optimal rehydration of the yeast. For this reason, none of the wine yeast manufacturer proposes the direct inoculation of active dried yeast to grape must. 
         [0005]    As known to the skilled person—in the present context the term fruit juice and fruit must are understood to be the same and these terms may herein be used interchangeably. 
         [0006]    As described in Soubeyrand et al. 2006—active-dried yeast (freeze dried) loose activity when not optimally rehydrated. The incorrect rehydration of active dried yeast can also lead to stuck alcoholic fermentation (O&#39;Kennedy, 2008). Therefore, the rehydration of active dried yeast needs a significant amount of time and concentration and usually demands a large number of skilled man-hours at a commercial winery during the winemaking process. 
         [0007]    EP1645198A1 (Minaki Advance Co Ltd, Japan); EP2090647A1 (Chr. Hansen); and WO2009/095137A1 (Chr. Hansen) may be considered as herein relevant prior art. However, none of these prior art documents discloses a method where yeast is added in frozen form to a fruit juice before fermentation. 
         [0008]    The present invention relates to a novel method for inoculating yeast into fruit juice or fruit, whereby the above mentioned drawbacks can be avoided. The inventors have found that, surprisingly, a frozen culture (frozen starter culture) of yeast can be directly added to grape juice while maintaining a high level of survival rate of the yeast cells. 
       SUMMARY OF THE INVENTION 
       [0009]    The present invention provides a new wine yeast product in a frozen form. The product is produced in a fermenter, concentrated, cryoprotectants are added. This mixture is then frozen at −50° C. in suitable containers. What makes this product unique is that besides the fact that it is frozen, is that it can be directly added to grape juice as no rehydration is required because the yeast was not dehydrated in the production process. The product is currently in the form of a frozen bag (plastic DIM bag) containing 1 kg of frozen yeast cell culture. 
         [0010]    As shown in Example 1 herein—when directly inoculating frozen yeast, to the surprise of the present inventors, only a relatively small decrease in cell numbers was observed compared to active dried yeast where a much larger decrease in cell numbers where observed when directly inoculated compared to correct rehydration. Therefore, this feature makes the frozen yeast suitable for direct inoculation, a simple process where an unskilled person can open the container and directly add the contents to grape juice, without risking significant cell death and consequent loss of activity, as is the case for active dried yeast. Furthermore, direct inoculation of yeast prevents potential spoilage of wine fermentations by contaminated rehydration mixtures. 
         [0011]    Accordingly, based on this surprising finding one can thereafter understand that the use of frozen yeast may be highly advantageous for wine production according to the present invention. 
         [0012]    A further advantage is that direct inoculation results in a significant time and man-power savings for winemakers as rehydration of active dried yeast can take up to an hour and needs a skilled person preparing highly specified rehydration substrates and direct inoculation of frozen yeast takes a few minutes and does not need special training or skills, simply adding the product to the grape juice or crushed grapes. 
         [0013]    The results provided herein have been obtained by using the wine yeast  Pichia kluyveri . The skilled person may easily transfer the technology to other types of yeast, such as  Saccharomyces cerevisiae, Saccharomyces pastorianus, Saccharomyces bayanus , or  Torulaspora delbreuckii, Kluyveromyces thermotolerans  etc. 
         [0014]    The product could also be applied to produce other beverages than wine such as beer, cider, sake, kefir, soft drinks and beverages where the action of the particular yeast is required and applied in a convenient, direct inoculation form. The starter culture may be used to ferment any type of sugar based medium, such as grape juice, or apple juice, or alternatively, whole fruits. 
     
    
     
       DRAWINGS 
         [0015]      FIGS. 1 and 2 : These figures show herein relevant experimental results as discussed in details in working Example 1 herein. 
       
    
    
     DETAILED DESCRIPTION 
       [0016]    In one aspect, the present invention relates to a method for inoculating a yeast into a fruit juice or fruit. Said yeast will cause a fermentation, and the product of this fermentation may be used in the food industry, or more specifically the beverage industry. Beverages which are contemplated are e.g. wine, beer, cider or other beverages which are produced by way of fermenting a fruit or vegetable material, such as fruit juice, vegetables, plants or fruit. Examples of fruit or vegetable material are grapes, grape juice, apples or apple juice, or barley. 
         [0017]    The yeast which is used may be any type of yeast which is used in the beverage or food industry. Examples include e.g.  Pichia kluyveri, Saccharomyces cerevisiae, Saccharomyces pastorianus, Saccharomyces bayanus, Torulaspora delbreuckii , or  Kluyveromyces thermotolerans  etc. 
         [0018]    It is common to use yeasts in the wine industry. Typical yeasts used in wine production are from the yeast family Saccharomycetaceae (ascomycetous yeasts). Yeasts from the genus  Saccharomyces  [e.g. the species  Saccharomyces cerevisiae  (SC)] are commonly used. Other used yeasts are from the same Saccharomycetaceae family but from other genera such as  Kluyveromyces  [e.g. the species  Kluyveromyces thermotolerans  (KT)] and the genus  Torulaspora  [e.g. the specie  Torulaspora delbrueckii  (TD)]. 
         [0019]    For the inoculation of fermenting a fruit or vegetable material, a pure yeast culture may be used (i.e. a culture containing only one type of yeast), but a mixed culture of two or more types of yeast may also be used as inoculant. 
         [0020]    For a mixed starter culture it may be important to know the specific amount/ratio of each yeast species in the final commercially available starter culture. This is in particular true for a wine starter culture comprising  Saccharomyces  ssp.,  Kluyveromyces  ssp. and/or  Torulaspora  ssp. 
         [0021]    The starter culture may be produced by fermenting yeast, followed by harvesting the yeast by centrifugation. This produces a liquid yeast biomass. Typically, a 1000 L fermenter produces about 100 kg of “wet weight yeast” in the form of a liquid paste the liquid paste. Based on wet weight, this paste typically contains approximately 10 10  CFU (colony forming units) per gram. After freezing, approximately 10 9  CFU per gram remains. 
         [0022]    To protect the cells from the harsh freezing conditions, a cryoprotectant may be added after or before centrifugation. Examples of cryopretectants include glucose, sucrose, trehalose. 
         [0023]    Such cryoprotectants are typically added in an amount of about 5%-20%. The amount should be sufficient to maintain the number of CFU at approximately 10 9  or above. 
         [0024]    After adding cryoprotectant and collecting the liquid yeast biomass, the biomass is transferred to suitable container, e.g. plastic bags. These containers are then frozen at a temperature of between −10° C. and −60° C. A preferred temperature interval is between −10° C. and −30° C. 
         [0025]    The method according to the invention entails adding the frozen starter culture to the sugar based medium in an amount which is sufficient to initiate and maintain fermentation of the vegetable material. 
         [0026]    Accordingly, an aspect of the invention relates to a method for inoculating yeast into fruit juice comprising the following steps:
       a) providing a fruit juice in a container; and   b) adding yeast in frozen form in an amount which is sufficient to initiate and maintain fermentation of the fruit juice.       
 
         [0029]    The skilled would in the present context understand that when you add yeast in frozen form to the fruit juice according to step b) above—there is no herein rehydration of the yeast. As understood by the skilled person—one may call the herein described method of the invention a direct inoculation method. 
         [0030]    In working Example 1 is discussed experiments relating to the use of frozen yeast starter cultures for inoculation. Surprisingly, it was found that by thawing the frozen yeast culture before it was added to the grape juice, the survival of the cells (CFU/ml) in the grape juice was even better than what was achieved when directly inoculating the frozen yeast culture in the grape juice. 
         [0031]    Accordingly, a separate aspect of the invention relates to a method for inoculating yeast into fruit juice comprising the following steps:
       i) providing a fruit juice in a container;   ii) providing yeast in frozen form;   iii) thawing the frozen yeast to get yeast in liquid form; and   iv) adding the yeast in liquid form in an amount which is sufficient to initiate and maintain fermentation of the fruit juice.       
 
         [0036]    The skilled person would in the present context understand that when one thaws the frozen yeast to get yeast in a liquid form and then add yeast in liquid form to the fruit juice according to step iii) above—there is no herein rehydration of the yeast. 
         [0037]    As known to the skilled person—the temperature for the fermentation of the fruit juice step in a winery is typically between 15 to 25° C. Accordingly, when one in a winery adds yeast in frozen form to the fruit juice [i.e. according to step b) above] then is the yeast frozen form typically thawed at a temperature between 15 to 25° C. 
         [0038]    In the working Example 1 herein was the thawing temperature 30° C. 
         [0039]    Without being limited to theory—it is believed that one gets the herein best results by the thawing temperature of step iii) above being from 25 to 35° C. 
         [0040]    As understood by the skilled person—the thawing of step iii) above may be done by e.g. adding the yeast in frozen form into e.g. water or a water juice mixture, wherein the water or a water juice mixture has a suitable temperature. As understood by the skilled person—one would thereby get yeast in liquid form that can be added to the fruit juice according to step iv) above. 
       EXAMPLES 
     Materials and Methods 
       [0041]    The active dried yeast used in this example was  Saccharomyces bayanus  B52 (Lesaffre). The frozen yeast used in this example was  Pichia kluyveri  (Chr. Hansen). All inoculations were performed in triplicate, with the standard error indicated on the bar graphs in  FIGS. 1 and 2  below. After inoculation with yeast, the mixture was agitated thoroughly to dissolve contents before making standard dilutions for plating. To determine the cell counts of these products, dilutions were made in peptone water and 1 ml poured into Petri-dishes after which Yeast Glucose Medium (YGM) agar was poured in liquid form at 40° C. Plates were incubated for 3 days at 30° C. after which colonies were counted. The CFU/g as indicated in  FIGS. 1 and 2  below was calculated based on the original weight addition of ADY and frozen yeast. To determine the cell counts of yeast after inoculation in synthetic grape juice medium, the same protocol was followed. An amount of 50 g/l of frozen or thawed  Pichia kluyveri  yeast was inoculated into 200 ml of synthetic grape juice medium at room temperature (21° C.). Active dried yeast was either rehydrated (20 min 37° C., 1 g in 10 ml of non-chlorinated water), or weighted out to perform direct inoculation with a 0.3 g/L, corresponding to 2*10 6  CFU/ml. For direct inoculation and rehydrated ADY, the temperature of the synthetic grape must was adjusted to 30° C. to facilitate greater survival due to the temperature difference of the inoculum and media. 
         [0042]    Synthetic grape must was prepared according to Costello et al. 2003 with a sugar concentration of 250 g/L and a pH of 3.5. After inoculation, dilutions were conducted in peptone water and 1 ml poured in Petri-dishes after which Yeast Glucose Medium (YGM) agar was poured in liquid form at 40° C. Plates were incubated for 3 days at 30° C. after which colonies were counted. Using the amount of yeast mass added to the synthetic grape must and the CFU/ml value obtained from the plate counts, a value in CFU/gram could be calculated. 
       Example 1 
     Use of Frozen Yeast—Comparison with Freeze-Dried Yeast 
       [0043]      FIG. 1  shows the results of the detrimental effect of direct inoculation of active dried yeast (ADY) when directly inoculated in synthetic grape juice (horizontal lines) compared to first rehydrating the ADY yeast in water at 37° C. for 20 minutes and then inoculating it in synthetic grape must (vertical lines). After inoculation, cell counts were conducted by pour plating and the data used to calculate an average CFU/g value. The CFU/g of the original active dried yeast was on calculated on average as 2.40E+10 (dotted). Direct inoculation of the active dried yeast resulted in calculated average CFU/g value of 4.49E+9 meaning that 81.3% of the cells died due to direct inoculation. However, when first rehydrated in water at 37° C. for 20 mins and then inoculated in synthetic grape juice, the calculated average CFU/g was 2.35E+10 meaning that only about 2.1% of the cells died after rehydration and inoculation. 
         [0044]      FIG. 2  shows the effect of direct inoculation of frozen yeast in synthetic grape must with and without thawing first. The cell count of the original frozen yeast product 8.0E+8 (dotted). After direct inoculation of frozen yeast (without thawing) in grape juice the cell count was calculated on average 5.1E+8 (horizontal lines) and after thawing then direct inoculating in grape juice the cell count was calculated on average 7.1E+8 (vertical lines) meaning that in the frozen direct inoculation (without thawing) only 36.3% of cells died and in the frozen direct inoculation (after thawing) only 11.3% of the cells died. This compares very favourably to the active dried yeast, where 81.3% of the cells died, indicating that frozen yeast can be used for direct inoculation without a significant loss in cell count and consequent activity. 
       CONCLUSIONS 
       [0045]    The results of this Example 1 indicate that active-dried yeast (freeze dried) looses a significant amount of viable cells when directly inoculated (without re-hydration) compared to inoculation after re-hydration in water (specific temperature) and juice dilution—i.e. these results were in agreement with the prior art knowledge as discussed above. 
         [0046]    However, when directly inoculated frozen yeast is used, to the surprise of the present inventors only a relatively small decrease in cell numbers was observed as in the case above. 
         [0047]    Accordingly, based of this surprising finding one can thereafter understand that use of frozen yeast may be highly advantageous for wine production according to the present invention. 
         [0048]    A further advantage is that direct inoculation results in a significant time saving for winemakers as rehydration of active dried yeast can take more than an hour and direct inoculation of frozen yeast takes a few minutes. 
         [0049]    As indicated in the example above, surprisingly, it has been found that by first thawing the frozen culture and then directly inoculating in grape juice, the survival of the cells (CFU/ml) was even better than what is achieved when directly inoculating the frozen yeast culture in grape juice without thawing first. 
       REFERENCES 
       [0000]    
       
         1. EP 1645 198 
         2. EP 2090 647 
         3. WO 2009/095137 
         4. Peter Costello, Paul Henschke and Andrew Markides (2003) Standardised methodology for testing malolactic bacteria and wine yeast compatibility. Australian Journal of Grape and Wine Research 9 (2): 127-137. 
         5. Roger Boulton, Vernon Singleton, Linda Bisson and Ralph Kunkee. 1996. Principles and Practices of Winemaking pp. 124. 
         6. Karien O&#39;Kennedy. (2008). How to avoid stuck fermentations, The Australian &amp; New Zealand Grapegrower &amp; Winemaker. November, Issue 538, 103-105. 
         7. Virginie Soubeyrand, Anne Julien and Jean-Marie Sablayrolles (2006) Rehydration Protocols for Active Dry Wine Yeasts and the Search for Early Indicators of Yeast Activity American Journal of Enolology and Viticulture. 57(4)-474-480.