AU2013206413A1 - Method for active dry yeast rehydration, and rehydration medium - Google Patents

Abstract

The invention concerns a method for rehydration active dry yeasts intended for 5 alcoholic fermentation. The method is characterised in it consists in placing that said active dry yeasts in an aqueous medium containing at least inactive yeasts or a yeast derivative, and optionally one or several complementary nutrients selected among sources of organic or inorganic nitrogen, vitamins, mineral salts, fatty acids, sterols, or natural sources rich in those elements. The invention also concerns the 10 rehydrating media used in said method. The invention which enables to avoid fermentation problems is useful for producing wines and other fermented alcoholic drinks.

Description

1 METHOD FOR ACTIVE DRY YEAST REHYDRATION, AND REHYDRATION MEDIUM The present invention concerns the technical sphere of 5 the production of alcoholic drinks, in particular that of the production of wines. In this sphere, problems associated with fermentation represent an important loss of profit in economic terms. In fact these fermentation problems translate, in the case of the 10 wines concerned, into output losses, loss of quality, indeed sometimes into volume losses. It is therefore important for businesses in this field to reduce the frequency of occurrence of fermentation problems. 15 In a manner which is now widespread, pure yeast cultures are used to permit a rapid start and regular progress of the fermentation of the grape musts. Pure cultures of vinification yeasts are stocks selected from the family of Saccharomyces belonging generally to 20 the species cerevisiae, uvarum or bavanus. However, one of the critical moments of fermentation remains the end of this fermentation. It is essential to be able to terminate this reaction with sufficient speed of consumption of sugars to avoid situations of 25 stoppages or of flagging fermentations which represent risk situations, when other micro-organisms, notably lactic acid bacteria are able in these circumstances to colonise rapidly the fermentation medium stopping definitively the alcoholic fermentation. Certain or 30 these micro-orgaanisms will utilise the residual sugars to produce undesirable metabolites such as, for example, acetic acid, resulting in losses of quality in the case of the wine concerned.

2 By problematic fermentations are to be understood fermentations corresponding to two types of situation: slow fermentations and flagging fermentation. The fermentation speed of a medium is defined as the 5 quantity of carbon dioxide released per time unit. It is represented by the curve derived from the quantity of carbon dioxide released as a function of time V=dCO 2 /dt. 10 In the case of slow fermentation, the maximum observed speeds in the course of the fermentation are low. These slow fermentations are generally attributable to yeast populations which have low metabolic activity, in general associated with a deficiency of assimilable 15 nitrogen in the musts. Flagging fermentations or fermentation stoppages are characterised by a maximum fermentation speed which is relatively high but which diminishes progressively, the 20 viability of the yeasts becoming very weak. These fermentations slow down significantly then or stop totally when the yeast population is insufficient to ensure the complete consumption of the sugars. In the majority of cases, this type of phenomenon is observed 25 when there is oxygen deficiency or major nitrogen deficiency. It is well known that these problematic fermentations are most frequently associated with imbalances or deficiencies of the nutritive media (Alexandre et al. 30 J. of Ind. Microbiol. and Biotechnology, vol. 20, 1998: pages 20-27) . Indeed, different nutrients are necessary LO allow yeasts on the one hand to develop sufficiently well to colonise the fermentation medium and on the other hand to ensure effectively the 3 metabolism of the sugars in alcohol, and this to the point of total exhaustion of these sugars. These nutrients belong especially to the following categories: sources of nitrogen, sources of oxygen, 5 vitamins, mineral salts. Assimilable nitrogen is constituted by ammonia nitrogen and o-amino-nitrogen. This is the nutrient which has the greatest influence on the speed of alcoholic fermentation (Agenbach. Proc. South African Soc. Enol. 10 Vitic., Cape town, South Africa. Stellenbosh SA, 1977: pages 66-87) . Nitrogen is an essential element since it allows the synthesis of protein, and thus the growth of the yeasts. Its deficiency in the must firstly limits the growth of yeast and thus the fermentation 15 speed and secondly causes a significant diminution in the movement' of sugars, thus increasing the risks of fermentation stoppage or of flagging fermentation. A nitrogen deficiency may also result in a deviation of the metabolism of the yeast, resulting especially in 20 the production of hydrogen sulphide

(H

2 S) , which is harmful to the aromatic characteristics of the wine. In general, musts contain between 80 and 400 mg/l while the deficiency threshold is situated between 150 and 180 mg/l. A nitrogen deficiency in the must is thus a 25 major risk for the good progress of wine production. Oxygen plays a paramount role in alcoholic fermentation. It ensures the proper development of the yeast population and supports the viability of the 30 yeast . It allows the synthesis of lipid compounds such as sterols or unsaturated fatty acids, constituting the cell membrane of the yeasts and fostering a better resistance to ethanol. The fluidity of the membrane is in effect a function of the ratio of unsaturated fatty 35 acids/saturated fatty acids. The presence of 4 unsaturated fatty acids is thus paramount for this resistance to alcohol. The minimum requirements of 02 to ensure the successful progress of a fermentation may be estimated at around 5 5 -10 mg/l (Sablayrolles and Barre. Sci. Alim., vol. 6, 1986: pages 373-383) . However, oxygen is very rapidly consumed at the start of fermentation firstly by the enzymes (case of decomposition: laccase), and secondly by the oxidising yeast flora. Wine growers are 10 therefore driven to add oxygen in the course of fermentation, before the medium is exhausted. The timing of the addition of this element (mid fermentation, after the multiplication of cells and the dilution of lipids in the fermentation flora) is thus 15 paramount. Another known means of avoiding fermentation problems linked to an oxygen deficiency is the addition to the must of unsaturated fatty acids, of sterols or of compounds rich in these molecules such as mud and 20 certain yeast derivatives. Certain vitamins play an essential role in the progress of alcoholic fermentation. Biotin for example encourages the production of esters and allows a better cellular viability at the end of the fermentation. In 25 the event of biotin deficiency, cellular growth is significantly affected. Pantothenate, involved in the metabolism of lipids, has a positive effect on the organoleptic qualities: it reduces the risks of production of H 2 S as well as of volatile acidity. 30 Thiamine finally, the role of which has been the subject of numerous studies, is another of the vitamins which can be limiting and the lack of which can cause stoppage of fermentatLon. This deficit is often attributable to certain pre-fermentation treatments 35 carried out in badly controlled conditions 5

(

s ulphidising, hea ing) . In fact, thiamine possesses the property of combining rapidly with

SO

2 and is then no longer biologically available. It can also moreover disappear rapidly from the medium. It has been 5 effectively shown (Bataillon et al., J. Ferm. Bioeng., vol. 82, 1996: pages 145-150) that in the presence of 106 cells of Saccharomyces cerevisiae /ml, virtually all of the thiamine has disappeared in 2 to 3 hours, the yeasts being able to accumulate important 10 concentrations of it. Moreover the non-yeast indigenous flora consumes this element sometimes more rapidly than the Saccharomyces cerevisiae. Other nutrients such as magnesium, zinc, manganese or potassium are also very important for the activity of 15 the yeast. Magnesium in particular plays an essential role in this control of cellular growth and the metabolism of the yeasts. It allows a better resistance to temperature and to osmotic pressure. It operates at the level of the maintenance of cellular 20 integrity (stabilisation of nucleic acids, of polysaccharides, lipids and proteins) (Walker, Critic. Rev. Biotechnol., vol. 14, 1994: pages 311-354). Magnesium additions increase the production of ethanol. Zinc is also very important for it is a co 25 factor of the enzymes in glycolysis; it allows a better alcohol tolerance and plays an important role in the production of esters. Even though deficiencies in these mineral elements are nfrepuenL, they are only rarely biologically 30 availabIe . In fact, as these cations can be linked to different types of molecules in rhe must such as proteins and polyphenols, the -east cannot use them. The composition of the musts in nutrients which are useful for the yeasts is thus considered a crucial 35 feature for the good management of wine production.

6 The possibility of a deficiency in one or other nutrient means taking an important risk in respect of the product which will ultimately be obtained. It is for this reason that certain solutions have been 5 proposed and are currently practised, in order to avoid these deficiency risks. Nowadays, a plurality of techniques are implemented in order to supplement deficient musts during the entire fermentation process and to reduce the frequency of 10 occurrence of problematic fermentations: - for deficiencies in nitrogen: the addition to the must of di-ammonia phosphate or of ammonium sulphate at mid- fermentation is advocated (Sablayrolles et al., J. Ferm. Bioeng., vol. 82, 1996: pages 377-381). 1s - for oxygen deficiencies and deficiencies in growth factors, the addition to the must of fine mud allows the control of turbidity and supplies nutrients such as sterols and unsaturated fatty acids (Delfini et al., Am. J. Enol. Vitic., vol. 44, 1993: pages 86-92). 20 However this addition is not always easy to implement in practice, in the cellars. It is for this reason that the addition of complex products containing inactive yeasts has been suggested in order to compensate for deficiencies in the must .

These 25 products generally contain, in addition to inactive yeasts or yeast hulls, ammonium phosphate and thiamine. Besides their nutritive properties, they influence the overall cruality of the wines. The effectiveness of the addition of such products has been demonstrated in 30 numerous oenological situations (Trioli. Vitic. Enol. Sci., vol. 51(3), 1996, pages 204-209). Recently, a particularly effective means of intervention has been developed in order to reduce the risks of fermentation stoppage (Sablayrolles et al., j.

7 Ferm. Bioeng. vol. 82, 1996: pages 377-381) . It consists in supplying simultaneously a nitrogen and oxygen to the must. It is then noted that the effects are cumulative. It is however paramount that these 5 combined additions are controlled; the timing of the supply and the quantity of nutrients supplied being essential. Finally the addition of thiamine is a current practice. Usually it is added at the start of fermentation which 10 renders it largely unavailable for the yeast responsible for ensuring alcoholic fermentation. It should be noted moreover, that recent studies on the characterisation of the needs, in terms of assimilable nitrogen and oxygen, of different fermentation yeasts I- allow the operator to choose his yeast according to the nutritive quality of the must which he has to ferment (Julien et al., Am. J. Enol. Vitic., vol. 51 (3), 2000: pages 215=222) . However this knowledge does not permit freedom from correcting the musts for certain nutritive 20 factors. Thus all the technical solutions described and utilised up to present are based on the same principle; realising one or more additions to the must of nutrients indispensable to the fermentative activity of 25 the yeast at the start of or in the course of fermentation. These methods do not make it possible to supply the useful nutrients directly to the yeast in a quantity and/or a concentration sufficient to maintain a high Lerm-en- * o L he a nd of the 30 fermentation speed right to e dhe Termentation, nor to escape the problem of the availability of certain elements essential to the good development of the fermentation yeast.

8 Consequently, despite the application of these techniques, there exist still a large number of situations of problematic fermentation. In the last twenty years, the use of specially selected 5 leavens in dried form (active dry yeasts, ADYs) has become widespread. Before they are used, it is necessary to operate a rehydration step. This ste is relatively simple to put into operation but is fundamental for the quality of the leaven once 10 rehydrated (Monk. Proceedings of the ASVO Seminar: Advances in Juice Clarification and Yeast Inoculation. Melbourne, Aus. 22-23, 1997). Numerous authors have provided the theoretical bases and have described the structural and biochemical 15 transformations which operate in yeast cells during dehydration and rehydration operations (M.J. Bakers et. al., Advances in Biochemical Engineering/Biotechnology, vol. 35, 1987, pp. 127-171) . The optimal conditions to be implemented during rehydration in order to avoid as 20 far as possible the destruction of the cells have also been the subject of various studies. One of the most complete was carried out by Radler et al. on the rehydration of vinification yeasts (Von F. Radler et al., Deutsche Lebensmittel-Rundschau, vol. 3, 1985, pp. 25 73-77) . It has thus been revealed that the composition of the rehydration medium had an influence on the metabolic activity of the yeast measured during the first two hours after rehydration. In particular the mixtures of grape juice and of water, and solutions 30 containing a specific quantity of glucose, fructose, malic acid, amino acids, 2-ketoglutaric acid, ammonium sulphate, vitamins or of KCl, of CaCl 2 and of NaCl have had a more or less marked effect on the metabolic activity of the dry yeast cells, the greatest increase 35 in activity having been obtained with a 1 % solution of KCl.

9 The present invention advantageously provides a very high rate of revivified cells due to defined rehydration conditions, the decisive criterion being the capacity of the yeast to resume high fermentation activity in the very short time limits. The composition of the rehydration media was designed as a function of this criterion, tested over tests of short duration, the nature of the 5 constituents of said media and the concentrations implemented aiming essentially at preserving the integrity of the cell membrane and stabilising the osmotic pressure, for example by the addition of salts. However, it had not previously been demonstrated that specific rehydration conditions of the yeast 10 could have a longer-term influence on the fermentation activity of the yeast cells. In particular, it had not previously been suggested that a solution permitting the reduction of problematic fermentation should be sought in the rehydration conditions of the yeasts, i.e. before they are introduced into the must to be fermented. 15 In a surprising and unexpected manner, it was found that rehydration of active dry yeasts (ADYs) in a medium with an added nutritive complex comprising inactive yeasts or yeast derivatives and including other nutrients permitted the fermentation capacity of the yeast produced from this rehydration step to be increased, especially at the end of fermentation, whereas it was not possible to observe such effects with the addition of this type of complex to the must. 20 Thus in one aspect, the present invention proposes a rehydration medium reducing problematic fermentation, suitable for the production by fermentation of wines and other alcoholic drinks. The subject matter of the present invention includes a medium for rehydrating active dry yeasts for 25 alcoholic fermentation wherein said medium comprises at least inactive yeast or a yeast derivative and one or more supplementary nutrients liable to be found in insufficient concentration or insufficient availability in the must. These supplementary nutritive elements are chosen from sources of organic or inorganic nitrogen, vitamins, mineral salts, fatty acids, sterols, or natural sources rich in these elements. 30 The active dry yeasts concerned are all vinification yeasts used to culture grape musts in order to insure good starting and regular progress of the fermentation. These are in particular yeasts of the Saccharomyces family, preferably Saccharomyces cerevisiae. 35 Inactive yeasts and yeast derivatives are well known to the person skilled in the art and are commercially available. It is understood that the present rehydration medium may comprise inactive 10 yeasts enriched with mineral salts, for example such as those described in the patent application GB 98 1110.0, or inactive yeasts enriched with any other nutrient. Yeast derivatives are all the products capable of being obtained from whole or partial yeast cells, by physical or chemical action. They include in particular, yeast extracts obtained by autolysis and yeast hulls. They present most often in 5 the form of more or less fine powder after grinding and according to the invention, they are used in suspension in the rehydration medium. The rehydration medium is based on an aqueous medium which may or may not be sugary. In the case of a sugary medium, glucose water or grape juice is used by preference, as is the current practice, .0 generally taken from the must which is intended to be fermented. The inactive yeasts or yeast derivatives as well as the supplementary nutrients are introduced into the base medium, then the ADYs are added in such a way that the rehydration takes place from the start in the medium as defined. [5 In a first aspect, the present invention provides an aqueous rehydration medium to rehydrate active dry yeasts used to ferment a grape must, comprising at least: i) inactive yeasts or a yeast derivative at the rate of 100 to 200 g/l of medium, and ii) one or several nutrients selected from the group consisting of: organic nitrogen sources of 20 ammonium salts, amino acids, peptides and proteins; fatty acids, sterols, a combination thereof or a natural source rich in these elements; vitamins; and mineral salts comprising zinc, magnesium and manganese salts or a combination of these salts; wherein each selected nutrient is provided at a concentration which is 200 to 1000 times greater than that of the said each selected nutrient within the grape must to be fennented. 25 In an embodiment, the medium is sugary. In an embodiment, the medium is not sugary. In an embodiment, each selected nutrient is added at a concentration which is about 500 times greater than that of the said each selected nutrient within a grape must to be fermented. 30 In an embodiment, the aqueous rehydration medium comprises at least: i) inactive yeasts or yeast extract, at the rate of 100 to 200 g/l of medium, and ii) one or several of the following nutrients: 0.2 mg/l of biotin, 50 mg/l of zinc salt, 4.33 mg/l of magnesium salt, and 4 mg/l of manganese salt. 35 11 In an embodiment, the aqueous medium is glucosed water at a rate of about 50 g/l or grape juice. In an embodiment, the medium comprises inactive yeasts or yeast extract at the rate of about 150 g/l of medium. 5 In a second aspect, the present invention provides a dry composition to prepare an aqueous rehydration medium to rehydrate active dry yeasts used to fennent a grape must, wherein the dry composition comprises about 74.85% by weight of inactive yeasts, about 25.0% di-ammonia phosphate, about 0.015% by weight of pantothenic acid, about 0.0085% by weight of thiamine, about 0.00012% by weight of biotin, about 0.015% by weight of zinc sulphate, about 0.11% by weight of 0 magnesium sulphate, and about 0.0011% by weight of manganese sulphate. In a third aspect, the present invention provides a dry composition to prepare an aqueous rehydration medium to rehydrate active dry yeasts used to ferment a grape must, wherein the dry composition comprises about 99.66% by weight of inactive yeasts, 0.018% by weight of calcium pantoth6nate, 5 0.00013% by weight of biotin, 0.033% by weight of zinc salt, 0.29% by weight of magnesium salt and 0.0027% by weight of manganese salt. In a fourth aspect, the present invention provides an aqueous rehydration medium prepared from the dry composition of the second or third aspects. .0 In particular, the rehydration medium for active dry yeast according to the invention can comprise at least i) inactive yeasts or yeast extracts, at the rate of 100 to 200 g/l, preferably 150 g/l, ii) one or more of the following consituents: 27 mg/i of calcium pantothenate, 0.2 mg/l of biotin, 25 50 mg/l of zinc sulphate, 433 mg/l of magnesium sulphate, 4 mg/1 of manganese sulphate, in an aqueous medium, preferably water with glucose added at a rate of 50 g/l, or grape juice. Another aspect of the present invention is a dry composition intended for the preparation of a rehydration medium for active dry yeasts as previously described, comprising at least 30 i) inactive yeasts or a yeast derivative, in dehydrated form, and ii) one or more supplementary nutrients, chosen from organic nitrogen sources of ammonium salts, amino acids, peptides, proteins, vitamins, mineral salts, fatty acids and sterols. In an embodiment, said dry composition can in particular contain 35 i) at least 98% by weight of inactive yeasts or a yeast extract, in dehydrated form, 12 ii) one or more nutrients chosen from ammonium salts, nitrates, urea, amino acids, peptides, proteins or a biological source rich in nitrogen; a fatty acid, a sterol, a combination of these or a natural source rich in these elements such as especially mud: thiamine, biotin, pantothenic acid, niacin, riboflavin, pyridoxine, or a natural source rich in vitamins; phosphates, salts of: zinc, 5 magnesium, calcium, potassium, sodium, iron, copper, manganese or a combination of these mineral salts. In an advantageous manner it can be made up of i) about 99.3% by weight of inactive yeasts or yeast derivative, in dehydrated form, L0 ii) one or more nutrients chosen from pantothenic acid, biotin, a zinc salt, a magnesium salt, a manganese salt. In a preferred embodiment, the present invention consists in a dry composition for the preparation of a rehydration medium for active dry yeasts comprising about 74.85% by weight of inactive yeasts, 15 about 25.0% di-ammonia phosphate, about 0.015% by weight of pantothenic acid, about 0.0085% by weight of thiamine, about 0.00012% by weight of biotin, about 0.015% by weight of zinc sulphate, about 0.11% by weight of magnesium sulphate, and about 0.0011% by weight of manganese sulphate. In a further preferred embodiment, the present invention consists in a dry composition for the 20 preparation of a rehydration medium for active dry yeasts comprising about 99.66% by weight of inactive yeasts, 0.018% by weight of calcium pantoth6nate, 0.00013% by weight of biotin, 0.033% by weight of zinc salt, 0.29% by weight of magnesium salt and 0.0027% by weight of manganese salt. In another aspect of the invention, the use of a dry composition for preparing a rehydration medium of 25 active dry yeasts, comprising at least inactive yeasts or yeast derivatives added to the rehydration medium at a rate of 100 to 200 g/l, preferably 150 g/l, under dehydrated form and one or several nutrients chosen from among organic nitrogen sources comprising ammonium salts, nitrates, urea, amino acids, peptides, proteins or a biological source rich in nitrogen; fatty acids, sterols, a combination thereof or a natural source rich in these elements such as especially mud; vitamins 30 comprising thiamine, biotin, pantothenic acid, niacin, riboflavin, pyridoxine, or a natural source rich in vitamins; mineral salts comprising phosphates, nitrates, zinc, magnesium, calcium, potassium, sodium, iron, copper, manganese salts or a combination of these mineral salts. The inoculum, prepared by rehydrating ADYs in the rehydration medium according to the invention 35 or with the aid of the claimed rehydration medium, can be used to culture a must intended for the production of a fermented alcoholic drink. It will be introduced into the must in a quantity defined in 13 advance as a function of the concentrations of ADY, inactive yeast or yeast derivatives and supplementary nutrients introduced into the rehydration medium according to the dilution criteria defined previously, i.e. from 200 to 1000 times, preferably 500 times. 5 The active dry yeasts are introduced into the rehydration medium thus prepared at a rate of 50 to 150 g/l, preferably 100 g per litre of medium; and finally the rehydration medium containing the active dry yeasts is rehydrated at a temperature of between 30' and 450, preferably 37' C, for 20 to 40 minutes, preferably 30 minutes. [0 The present invention will find natural application in the production of a fermented alcoholic drink from a grape juice, such as a wine. A fermented alcoholic drink thus obtained is also claimed. The following examples illustrate in a non-restrictive manner embodiments of the present invention. 15 EXAMPLES Example 1: Improvement of the Fermentation Profile of a Synthetic must by the Incorporation of a Yeast Extract in the Rehydration Medium of the Fermentation Yeast. Three fermentations were carried out in parallel and in duplicate on a synthetic must MS70-fa (such 20 as described in Bely et al. (1990)) corresponding to a medium deficient in assimilable nitrogen (100 mg/I) and 25 14 containing 200 g/l of fermentable sugars. The active dry yeast used is the commercial yeast Lalvin EC1118. Rehydration media Mo: control rehydration medium: water with glucose 5 added at a rate of 50g/l. Me: rehydration medium containing a yeast extract (Bacto-yeast extract ref. 0127-01-7, DIFCO, USA) , used in a dosage such as to permit a final concentration of extract in the fermentation must of 30 g/hl, or 150 g 10 per litre of rehydration medium. Rehydrat ion 1 - One gram of the active dry yeast EC 1 1 1 8 0T is rehydrated in 10 ml of medium Mo at 370C for 30 minutes. 15 2 - One gram of the active dry yeast Lalvin EC 1 1 8 TM i rehydrated in 10 ml of medium Me at 37'C for 30 minutes. Fermentations Fo: control fermentation carried out with the yeast 20 rehydrated in medium Mo. No yeast extract is present. Fl: fermentation carried out with the yeast rehydrated in medium Mo. A yeast extract is added at the start of the alcoholic fermentation directly into the synthetic must at a rate of 30g/hl. 25 F2: fermentation carried out with the yeast rehydrated in medium Me containing the yeast extract which is to be found at a level of 30g/hl in the synthetic must.

15 Three fermentors containing 1.1 litres of synthetic must MS70-fa are inoculated with 2.2 ml of rehydration solution (which corresponds to a utilisation dosage of 20 g/hl of active dry yeast) . The fermentation 5 temperature is 24C. The production of CO2 is increased as a function of time. The results obtained (figure 1) show that the fermentation carried out by the rehydrated yeast in the presence of yeast extract in the rehydration medium 10 (form 2) terminates before the two other fermentations (forms Fo and Fl) . The kinetics of the end of fermentation characterised by the slope of the curve are more rapid in form F2 explaining the observed time saving of approximately 50 hours in comparison with the 15 other forms. The addition of yeast extract, at the start of aliboholic fermentation, does not, in these conditions, permit an improvement in the fermentation profile in comparison with the control form Fo. Example 2: Improvement of the fermentation profile of a 20 real Chardonnay must by the incorporation of a yeast extract into the rehydration medium of the fermentation yeast Two fermentations are carried out in parallel and in duplicate on a real Chardonnay must coming from the 25 south of France (Languedoc region) and containing 365 mg/l of assimilable nitrogen (situation of no deficiency) and containing 220 g/l of fermentable sugars. The active dry yeast used is the commercial yeast Lalvin 2C 1 1 1 8 TM, inoculated in a dosage of 20 30 g/hl. Rehydration One gram of the dry yeast Lalvin

EC

11 1 8 TM Is rehydrated in 10 m 1 of- glucose water (50 g/l) at 37C for 30 35 minutes. Tn the control rehydration, no addition is 16 made. In form 2, 1. 5 g of yeast extract (Bacto-yeast extract ref. 0127-01-7, DIFCO, USA) is added to 10 ml of the rehydration medium. Permentation The two fermentations correspond to the following forms: Fl: Fermentation carried out with the yeast EC11 1 8 TM rehydrated in a rehydration medium in the absence of 10 yeast extract. The yeast extract is added at the start of alcoholic fermentation directly into the Chardonnay must in a dosage of 30 g/hl. F2: Fermentation carried out with the yeast EC 1 1 1 8T rehydrated in the presence of a yeast extract (used in 15 a dosage permitting a concentration of 30 g/hl in the must) in the rehydration medium. The fermentors of 1.1 litres are inoculated with 2.2 ml of rehydration solution containing the ADYs, corresponding to a utilisation dosage in the must of 20 20 g/hl of active dry yeasts. The fermentation temperature is 24'C. The production of CO 2 increases as a function of time. The results obtained (fig. 2) show that the fermentation carried out by the rehydrated yeast in the 25 presence of yeast extract in the rehydration medium (form F2) is completed before the control fermentation F1 . The kinetics of the end of fermentation characterised by the sope of the curve are more rapid in form F2 exoIaining the 'time saving observed in 30 comparison with the control form. Even in the absence of a deficiency of assimilable nitrogen, the addition of yeast extract, during rehydration, permIts an improvement in the end of alcoholic fermentation by influencing the fermentation kinetics (420 slope of the 17 curve of end of fermentation by comparison with 260 in the control fermentation) . The result of exampQle 1 observed in the synthetic must is thus confirmed with the real must, namely that the addition of yeast 5 extract into the rehydration medium allows a faster fermentation than with the same addition in the must at the start of alcoholic fermentation. Example 3: influence of the addition of different nutrients on the duration of fermentation of a 10 deficient synthetic must 12 fermentations were carried out in parallel and in duplicate on a synthetic must MS70-fa such as described in example 1, and corresponding to a medium deficient in assimilable nitrogen (100 mg/l) and 15 containing 2'00 g/l of fermentable sugars. The active dry yeast used is the commercial yeast Uvaferm CEGTM inoculated at a dosage of 20 g/hl. The yeast extract is the commercial extract Bacto-yeast extract ref. 0127= 01-7 (DIFCO, USA). The vitamins and mineral salts are 20 the standard products available from suppliers. The 12 fermentations correspond to the following forms: F1 and 2: Controls in which no yeast extract is added either into the rehydration medium of the yeast or into 25 the fermentation medium. The control rehydration medium is the base medium: water with glucose added at 50 g/l. F3:) Fermentation carried out with the yeast CEG M rehydrated in the presence of yeast extract (used at a 30 dosage permitting a concentration of 30 g/hl in the must) in the rehydration medium. F4: Fermentation carried out with the yeast CEGIM rehydrated in a control rehydration medium. The same 18 yeast extract as for F3 is added at the start of alcoholic fermentation directly into the synthetic must MS70-fa in a dosage of 30 g/hl. F5: Fermentation carried out with the yeast CEGTM 5 rehydrated in the presence of inactive yeast (LBI2130TM Lallemand, Canada, utilised in a dosage permitting a concentration in the must of 30 g/hl) n the rehydration medium. F6: Fermentation carried out with the yeast CEGIM 10 rehydrated in a control rehydration medium. The same inactive yeast as for F5 is added at the start of alcoholic fermentation directly into the synthetic must MS70-fa in a dosage of 30 g/hl. F7: Fermen'tation carried out with the yeast CEGTM 15 rehydrated in the presence of di-ammonia phosphate (used in a dosage permitting a concentration of 10 g/hl in the must) in the rehydration medium. F8: Fermentation carried out with the yeast CEGT rehydrated in the control rehydration medium. Di 20 ammonia phosphate is added at the start of alcoholic fermentation in a dosage of 10 g/hl. F9: Fermentation carried out with the yeast CEGTM rehydrated in the presence of a cocktail of vitamins (pantothenate, thiamine and biotin) in the rehydration 25 medium in such a fashion as to obtain respective concentrations in the must of 60 ug/l, 34 ig/1, and 0.5 F10: Fermentation carried out with the yeast CEGM rehydrated in the control rehydration medium. The 30 cocktail of vitamins used for F9 is added at the start of alcoholic Fermentation at the concentrations indicated above.

19 Fli: Fermentation carried out with the yeast CEG TM rehydrate in the presence of a cocktail of mineral salts (Mg 2 +, Zn 2 , Mn 2 in the form of sulphates) in the rehydration medium in such a way as to obtain 5 respective concentrations of the salts in the must of 460 pLg/l, 58 g/l and 4.5 pg/l. F12: Fermentation carried out with the yeast CEGTM rehydrated in the rehydration medium. The cocktail of mineral salts used for Fil is added at the start of 10 alcoholic fermentation at the concentrations indicated above. The rehydration conditions are identical whatever the type of added product (yeast extract, inactive yeast, diammonia phosphate, cocktail of vitamins or of mineral 15 salts). The' correspond to those applied in examples 1 and 2. The volumes and conditions of fermentation are identical to those described in examples 1 and 2. The results obtained are recorded in table 1 below. The values presented correspond to the average duration of 20 fermentation in hours with duplicates for each form. Table 1 fermentation time, in hours 25 Added product in rehydration at start of medium fermentation 0 290 290 Yeast extract 250 280 Tnactive yeast 275 282 30 Di-ammonia phosphate 280 285 Cocktail of vitamins 283 288 Cocktail of salts 265 270 20 The results show that no matter what type of nutritive element is added, the additions during the rehydration phase result in a better reduction in the duration of fermentation than the same additions made at the start 5 of alcoholic fermentation. Example 4: Dry composition and corresponding rehydration medium Ml Dry powder composition ready for use: 10 inactive yeast 998.10 g di-ammonia phosphate 333.33 g pantothenic acid 200.00 mg thiamine 113.33 mg biotin 1.65 mg 15 zinc sulphate 195.00 mg magnesium sulphate 1500.00 mg manganese sulphate 15.00 mg A quantity of 150 g of this dry composition is weighed 20 and introduced into 1 litre of water with 50g/l glucose added. The following rehydration medium M1 is obtained. inactive yeast 149.71 g/l di-ammonia phosphate 50.00 g/l 25 pantothenic acid 30.00 mg/l thiamine 17.00 mg/l biot in 0.25 mg/l zinc sulphate 29.25 mg/I magnesium sulphate 225.00 mg/1 30 manganese sulphate 2.25 mg/1 Example 5: Method of rehydrating a commercial vinification yeast 21 15 g of dry active yeast Lalvin EC1118T are introdu ced into 100 ml of rehydration medium Ml prepared according to example 4. After incubation for 3 minutes at 3 ,j 5 an inoculum is obtained which i s ready to culture a must at the rate of 20 ml of inoculum per litre. The concentrations of nutrients supplied to the must are hus the following (it is assumed that the inoculated volume contains the said elements either in free form 10 in the aqueous phase of the rehydration medium, or in the form assimilated by the rehydrated yeasts) di-aLmonia phosphate 0.10 g/l pantothenic acid 60.00 pg/l 15 thiamine 34.00 pg/l biotin 0.50 !g/1 zinc salt 0.058 pg/l magnesium salt 0.45 tg/l manganese salt 4.50 ug/l 20 Example 6: Rehydration medium M2 and composiLion C2 In one litre of purified water are mixed 50 g of glucose and 150 g of the dry composition C2, 25 comprising: inactive yeasts 149.49 g calcium pantotnenate 27.0 mg biotin 0.2 mg zinc salt 50.0 mg magnesium salt 4.33 mg 30 manganese salt 4.0 mg -n this medium 12, 100 g of active dry yeast Uvaferm CECT, marketed by Lallermand, will be rehydrated. The 22 medium M2 containing the rehydrated yeast will be inoculated into a must to be fermented at a rate of 0.08 to 0.4 l/hl, preferably 0.2 1/hl. 5 Example 7: Application to the preparation of a fermented drink The medium M2 containing the composition C2 has been used to rehydrate the ADYs and culture a fermentation 10 must of a white wine in industrial conditions in a cellar in the region of la Mancha in Spain. Type of vine: Airen Assimilable nitrogen in the must: 225 mg/l (non deficient must) 15 Fermentations have been carried out in 100 hectolitre vats according to different forms, in order to determine the most favourable conditions. Two types of ADY have been used, on their own or in the presence of 20 other substances jointly added to the musts to encourage fermentation. In particular the influence of diammonium phosphate and a fermentation activator, added to the fermentation medium, has been studied. 25 The active dry yeasts UvafermTM CEG (Lallemand, Canada) MTM Mhv and Uvaferm PM (Lallemand, Canada) have been rehydrated with the aid of the medium M2 described in example 6. For each of the two yeasts, 4 tests have been carried out, by adding: 30 Test no. 1 Di-ammonia phosphate 0.2g/1 start of fermentation Test no. 2 Di-ammaonia phosphate 0.2g/l start of fermentation Fermentation activator 0.3g/1 first 1/3 fermentation 35 23 Test no. 3 Composition C2 0.3g/1(equiv) rehydration medium Di-ammonia phosphate 0.2g/1 start of fermentation Test no. 4 Composition C2 0.3g/1(equiv) rehydration medium 5 Fermentation activator 0.3g/l first 1/3 fermentation The fermentation activator used is the activator Fermaid T (Lallemand, Canada). 10 The kinetic profile of these eight fermentations was followed by measuring the density of the must and at the level of the production of a secondary component of fermentation, i.e. acetate. Indeed, acetate is responsible for the volatile acidity of wines which 15 should be minimised in order to obtain good gustative qualities. The results obtained are recorded in tables 2 and 3 below. The values presented in table 2 correspond to 20 fermentation times in days for each form. Table 2 fermentation time, in days 25 Test no. Uvaferm TM CEG Uvaferm-"PM 10 8 2 9 7 30 3 9 7 4 9 6 It appears that for the two active dry yeasts tested, the addition of the composition C2 into the rehydration 35 medium makes it possible to substantially improve the 24 profile of the fermentation kinetics (Test no. 3 compared to Test no. 1). Moreover, as can be seen with Test no. 4 in which the most rapid kinetics are obtained, the effect obtained by the addition of the 5 composition C2 into the rehydration medium for the ADYs acts in a cumulative manner with the effect produced by the addition of a fermentation activator. The values presented in table 3 correspond to the 10 volatile acidity measured at the end of fermentation by the Duclaux-Gayon method, known to the person skilled in the art. Table 3 15 Volatile acidity, in g/l Test no. Uvaferm TM CEG UvafermM PM 20 1 0.48 0.33 2 0.51 0.33 3 0.44 0.18 4 0.46 0.22 25 Tn view of these results, it appears that the addition of the composition C2 during rehydration makes it possible to reduce the production of volatile acidity. This could be attributable to the fact that the yeasts are in better physiological condition and thus capable 30 of better withstanding the physico-chemical conditions encountered in a must which is fermenting. This physiological state would then translate into a better resistance to stress, causing a smaller production of volatile acidity.

25 This supplementary positive effect reinforces the interest of the method according to the present invention for the rehydration of ADYs intended for alcoholic fermentation. Generally speaking, the results obtained at the end of these tests confirm this interest on an industrial scale. 5 Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. 10 All publications mentioned in this specification are herein incorporated by reference. Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is solely for the purpose of providing a context for the present invention. It is not to be taken as an admission that any or all 15 of these matters form part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia or elsewhere before the priority date of each claim of this application. It will be appreciated by persons skilled in the art that numerous variations and/or 20 modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive. 25

Claims (13)

1. An aqueous rehydration medium to rehydrate active dry yeasts used to ferment a grape must, comprising at least: i) inactive yeasts or a yeast derivative at the rate of 100 to 200 g/l of medium, and ii) one or several nutrients selected from the group consisting of: organic nitrogen sources of ammonium salts, amino acids, peptides and proteins; fatty acids, sterols, a combination thereof or a natural source rich in these elements; vitamins; and mineral salts comprising zinc, magnesium, and manganese salts or a combination of these salts; wherein each selected D nutrient is provided at a concentration which is 200 to 1000 times greater than that of the said each selected nutrient within the grape must to be fermented.

2. The aqueous rehydration medium of claim 1, wherein the medium is sugary. 5

3. The aqueous rehydration medium of claim 1, wherein the medium is not sugary.

4. The aqueous rehydration medium of any one of claims 1 to 3, wherein each selected nutrient is added at a concentration which is about 500 times greater than that of the said each selected nutrient within a grape must to be fermented. 0

5. The aqueous rehydration medium of claim 1, comprising at least: i) inactive yeasts or yeast extract, at the rate of 100 to 200 g/l of medium, and ii) one or several of the following nutrients: 0.2 mg/l of biotin, 50 mg/l of zinc salt, 4.33 mg/l of magnesium salt, and 4 mg/l of manganese salt. 5

6. The aqueous rehydration medium of claim 5, wherein the aqueous medium is glucosed water at a rate of about 50 g/l or grape juice.

7. The aqueous rehydration medium of any one of claims 1 to 6, wherein the medium comprises 30 inactive yeasts or yeast extract at the rate of about 150 g/l of medium.

8. A dry composition to prepare an aqueous rehydration medium to rehydrate active dry yeasts used to ferment a grape must, wherein the dry composition comprises i) inactive yeasts or a yeast derivative, in dehydrated form, and 35 ii) one or more supplementary nutrients, chosen from organic nitrogen sources of ammonium salts, amino acids, peptides, proteins, vitamins, mineral salts, fatty acids, and sterols. 27

9. A dry composition to prepare an aqueous rehydration medium to rehydrate active dry yeasts used to ferment a grape must, wherein the dry composition comprises i) at least 98% by weight of inactive yeasts or a yeast extract, in dehydrated form, ii) one or more nutrients chosen from ammonium salts, nitrates, urea, amino acids, peptides, 5 proteins or a biological source rich in nitrogen; a fatty acid, a sterol, a combination of these or a natural source rich in these elements such as especially mud: thiamine, biotin, pantothenic acid, niacin, riboflavin, pyridoxine, or a natural source rich in vitamins; phosphates, salts of: zinc, magnesium, calcium, potassium, sodium, iron, copper, manganese or a combination of these mineral salts. 0

10. A dry composition to prepare an aqueous rehydration medium to rehydrate active dry yeasts used to ferment a grape must, wherein the dry composition comprises i) about 99.3% by weight of inactive yeasts or yeast derivative, in dehydrated form, ii) one or more nutrients chosen from pantothenic acid, biotin, a zinc salt, a magnesium salt, a manganese salt. :15

11. A dry composition to prepare an aqueous rehydration medium to rehydrate active dry yeasts used to ferment a grape must, wherein the dry composition comprises about 74.85% by weight of inactive yeasts, about 25.0% di-ammonia phosphate, about 0.015% by weight of pantothenic acid, about 0.0085% by weight of thiamine, about 0.00012% by weight of biotin, about 0.015% by weight 20 of zinc sulphate, about 0.11% by weight of magnesium sulphate, and about 0.0011% by weight of manganese sulphate.

12. A dry composition to prepare an aqueous rehydration medium to rehydrate active dry yeasts used to ferment a grape must, wherein the dry composition comprises about 99.66% by weight of 25 inactive yeasts, 0.018% by weight of calcium pantothenate, 0.00013% by weight of biotin, 0.033% by weight of zinc salt, 0.29% by weight of magnesium salt and 0.0027% by weight of manganese salt.

13. An aqueous rehydration medium prepared from the dry composition of any one of claims 8 to 12.