EP1814978A2 - Means for improving fermenting capacities of active yeasts and uses thereof - Google Patents

Means for improving fermenting capacities of active yeasts and uses thereof

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Publication number
EP1814978A2
EP1814978A2 EP05818158A EP05818158A EP1814978A2 EP 1814978 A2 EP1814978 A2 EP 1814978A2 EP 05818158 A EP05818158 A EP 05818158A EP 05818158 A EP05818158 A EP 05818158A EP 1814978 A2 EP1814978 A2 EP 1814978A2
Authority
EP
European Patent Office
Prior art keywords
yeasts
fermentation
sterol
sterols
active
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP05818158A
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German (de)
French (fr)
Inventor
Jean-Michel Salmon
Anne Ortiz-Julien
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Danstar Ferment AG
Institut National de la Recherche Agronomique INRA
Original Assignee
Institut National de la Recherche Agronomique INRA
Lallemand SA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Institut National de la Recherche Agronomique INRA, Lallemand SA filed Critical Institut National de la Recherche Agronomique INRA
Publication of EP1814978A2 publication Critical patent/EP1814978A2/en
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • 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
    • C12N1/04—Preserving or maintaining viable microorganisms

Definitions

  • the invention relates to means for improving the fermentative capacities of active yeasts. It more particularly relates to active dry yeasts (LSA abbreviated) or active yeasts rehydrated, high sterol content and obtaining them.
  • LSA active dry yeasts
  • the invention also relates to the applications of these yeasts in alcoholic fermentation processes, in particular for the manufacture of fermented alcoholic beverages.
  • Sterols are important constituents of the yeast cell membrane.
  • concentration of sterols should be optimal to ensure membrane integrity, guaranteeing the activity of membrane proteins, such as permeases.
  • the sterol composition of yeasts therefore plays a major role in their fermentative capacity.
  • inactivated yeasts with a high content of certain types of sterols that is, naturally, either by enrichment, or by using sterol compositions in soluble form. It has indeed been found that the addition of such yeasts or compositions in the rehydration medium of active yeasts or active yeasts in a fermenting wort makes it possible to stabilize the sterols and to have soluble forms capable of being used. incorporated effectively by yeasts.
  • the fermentative capacities of the active yeasts of the fermentation medium are particularly high, especially during the stationary phase of the fermentation, the duration of the fermentation being generally shorter.
  • the invention therefore relates to active dry yeasts or rehydrated active yeasts with improved fermentation capacities. It also aims at a method of easy implementation for obtaining them.
  • the invention also aims to take advantage of the fermentative properties of these active yeasts for the manufacture of alcoholic fermented beverages, in particular wines, beers and strong alcohols such as vodka or whiskey.
  • the invention thus provides LSAs that can be used in rehydration and / or alcoholic fermentation, or rehydrated active yeasts that can be used in fermentation, or yeasts that can be used in fermentation, having a sterol content having at least two conjugated double bonds at the B-ring level. at least 200 ⁇ g / g dry weight, and more particularly at least 900 ⁇ g / g dry weight.
  • yeasts usable in fermentation means the yeast cream from their production or frozen yeasts.
  • yeasts or brewer yeasts or yeasts of distillery, which includes yeasts of the genus Saccchromyces or non Saccchromyces genus.
  • Saccharomyces belonging to the cerevisiae, uvarum and bayamis species will be mentioned.
  • these micelles comprise at least 10 to 20 mg / g (dry weight) of sterols, in particular at least 15 mg / g (dry weight), and approximately 600 to 800 mg / g (dry weight) of lipopolysaccharide-protein complexes. , precipitated with 80% (v / v) ethanol, in particular 660 to 670 mg / g (dry weight), with an average molecular weight of 160 to
  • the sterols comprise at least 20-25% ergosterol and 20-25% zymosterol, other sterols being present, such as ergosterol, lanosterol or ergosta 5 - 7 dienols, without this enumeration being limiting.
  • the sterol-enriched LSAs are obtained by addition in their rehydration medium or in the fermentation mash of inactivated yeasts, themselves having a sterol content having at least two conjugated double bonds at the same time.
  • B cycle level of at least 200 ⁇ g / g dry weight, and more particularly at least 900 ⁇ g / g (dry weight).
  • Such inactivated yeasts are yeasts naturally rich in sterols or yeasts enriched in sterols, in particular ergosterol, and / or zymosterol, and / or ergosta 5-7 dienols, advantageously according to the techniques known to those skilled in the art.
  • the yeasts used advantageously have a sterol content having two conjugated double bonds in ring B, from 900 to 1250 ⁇ g / g dry weight.
  • the amounts of inactivated yeasts added to the rehydration medium are of the order of 20 to 500 g / l, preferably 100 to 200 g / l, in particular of the order of 150 g / l.
  • LSA is enriched by transferring sterols in the form of micelles from inactivated yeasts.
  • the yeast sterol enrichment is obtained by addition to LSA in their rehydration medium or to LSA. added to fermentation mashes, a lipid composition containing a sterol fraction comprising ergosterol and zymosterol and a complex of lipopolysaccharides - proteins precipitable with ethanol.
  • compositions advantageously contain from 10 to 70% by dry weight of ergosterol and from 10 to 70% by dry weight of zymosterol, in a proportion of 10 to 20 mg of total sterols per g. and from 500 to 1000 mg / g of lipopolysaccharide-protein complex.
  • the sterol fraction further comprises one or more sterols such as fecosterol, lanosterol, ergosta 5-7 dienols (dihydroergosterol, methylzymosterol, desmethyllanosterol, and ergosta-7,22-dien-3-ol).
  • sterols such as fecosterol, lanosterol, ergosta 5-7 dienols (dihydroergosterol, methylzymosterol, desmethyllanosterol, and ergosta-7,22-dien-3-ol).
  • lipid compositions as used in the above process are novel products and, as such, are also within the scope of the invention.
  • compositions have in particular the following physicochemical properties:
  • micellar aggregates with an average diameter of approximately 380 nm (value obtained by measuring the diffraction of laser light) by PCS technique (Photo Correlation Spetroscopy).
  • the above compositions are used in a proportion of 1.5 to 3 g / l, preferably 2 g / l of rehydration medium or 20 to 100 g / hectare of must. fermentation.
  • Yeasts with a high sterol content as obtained by enrichment during their rehydration or fermentation phase, through the transfer and incorporation of Sterols in the form of micelles from inactivated yeasts, or by incorporation of said lipid compositions, are of great interest in fermentative processes in view of their high fermentation capacities during the stationary phase of fermentations.
  • this high sterol content decreases the production of volatile acids, which improves the organoleptic properties of the fermentation products.
  • the invention thus also relates to the use, as processing aids, of inactivated yeasts rich in sterols, as defined above, in LSA rehydration media or in alcoholic fermentation media, for the production of alcoholic fermented beverages, especially wines or beers, or alcohols whiskey, vodka and others.
  • the invention also relates to a process for producing such beverages comprising the addition of sterol-enriched active dry yeasts as defined above, on the basis of
  • yeasts are either yeasts naturally rich in sterols, rehydrated or not, or yeasts enriched as defined above, namely rehydrated active yeasts or LSA.
  • FIG. 1 rehydration of an LSA in the presence of sterol preparations according to the invention, with different contents of sterols; and - Figure 2, the incorporation of sterols by LSA during a rehydration phase using a sterol preparation according to the invention.
  • a supernatant 1 and a pellet L are obtained.
  • the supernatant is subjected to an ultracentrifugation step at 85,000 g, 3h, at 4 ° C.
  • a pellet 2, a supernatant 2 and a white colloid are obtained on the surface.
  • the colloidal fraction white is lyophilized to give the stabilized sterol preparation in soluble form (about 20 mg).
  • Active dry yeast Ig LALVIN ECl 118 ® is rehydrated in 10ml of a rehydration medium consisting of water with 50g / l of glucose. Rehydration is
  • FIG. 1 The examination of this figure shows that the fermentations conducted by the rehydrated yeasts in the presence of the sterol formulation used end before the control fermentations.
  • EXAMPLE 3 Effect of Formulation of Sterols According to Example 1 on the Activation of the Incorporation of Sterols by LSAs
  • the procedure is as in Example 1, but the rehydration is carried out in the presence of 2 ⁇ l of [4] - 14 C] cholesterol (50-60 mCi / mMol (Amersham CFA 128)), solubilized in 20 m ⁇ l of tergitol / ethanol mixture (v / v) in the presence or absence of 24 mg of the sterol formulation of Example 1 for the manufacture of alcoholic fermented beverages.
  • the radioactivity incorporated over time by the LSAs is measured as follows:
  • the yeast strains are grown under constant and strong aeration.
  • the temperature of the propagation is of the order of 15 to 40 ° C, preferably 25 to 35 ° C.
  • Yeast culture is carried out by constant supply of carbon sources and nitrogen sources, as well as intermittent input of essential growth factors.
  • the carbon sources are generally derived from molasses and may be molasses of sugar, beet or their mixture in varying concentrations.
  • the nitrogen source can come from various ammonia derivatives, such as ammonia hydroxide, ammonium chloride, ammonium sulfate, ammonium phosphate, di-ammonium phosphate or crude protein extracts .
  • the propagation of the yeast is carried out according to a program comprising variations in the growth rates.
  • the maximum growth rates are from 0.05 / h to 0.25 / h, preferably from 0.15 to 0.2 / h.
  • Enrichment of yeast sterols can be done by specifically modifying the carbon and nitrogen sources to reduce the total nitrogen rate relative to that of carbon.
  • sterol precursor sources include ethanol, acetic acid, squalene or lanosterol. These precursors may be added in purified or crude form. -.
  • compositions are obtained by a process comprising the following steps: incubation with stirring of a suspension in water of inactivated oenological yeasts under conditions making it possible to obtain micelles formed of fractions specifically enriched with sterols incorporated into complexes of lipids - proteins,
  • micellar fractions separation of the micellar fractions, followed by freeze-drying.
  • the separation step, after the incubation, of the micellar fractions is more particularly carried out by centrifugation.
  • the collected supernatant is then subjected to an ultracentrifugation step to increase the enrichment of the sterol micelle fraction, the supernatant colloid fraction is isolated and lyophilized.
  • compositions obtained contain a fraction of sterols composed of 10 to 70%, especially 20 to 25% by dry weight of ergosterol, of 10 to 70%, especially 20 to 25% by dry weight of zymosterol in a proportion of 10 to 20 mg of total sterols per g, and a complex of 80% (v / v) ethanol-precipitable lipopolysaccharide proteins, with an average molecular weight of about 160-170 kDa, at 600 to 800 mg /boy Wut.
  • the sterol fraction further comprises one or more sterols, such as fecosterol, lanosterol, and ergostasterols 5-7 dienols.
  • sterols such as fecosterol, lanosterol, and ergostasterols 5-7 dienols.
  • the polysaccharides, the lipids and the proteins are advantageously present on the basis of
  • Preferred polysaccharides have an average MW of about 30-35 kDa, especially 30-32 kDa, and contain mannose of glucose and inositol. Appropriate proportions respectively correspond to
  • the lipid fraction comprises, in varying amounts, preferably Cl 7: 1, C 16: 0 and Cl 8: 0 fatty acids. This lipid fraction is bound to the polysaccharides via a phosphatidylinositol group.
  • the remainder of the preparation is 80% (v / v) ethanol soluble membrane fragments.
  • the micelles formed are for example composed of: 1) At least 15 mg / g (dry weight) of sterols, composed of at least 20-25% of ergosterol, 20-25% of zymosterol and varying amounts of other sterols (fecosterol, lanosterol, ergosta 5- 7 dienols, etc.). 2) 667 mg / g (dry weight) of 80% (v / v) ethanol-precipitable lipopolysaccharide-protein complexes, with an average molecular weight of 167 kDa, composed of 27% of polysaccharides, 61% of lipids by weight and 12% of proteins.
  • the polysaccharides have an average molecular weight of 31.7 kDa, and contain 61% mannose, 33% glucose and 0.9% inositol.
  • the lipid fraction is formed of C17: 1, C16: 0 and C18: 0 fatty acids in varying amounts.
  • the lipid fraction is bound to the polysaccharides via a phosphatidyl inositol anchor. 3) 333 mg / g (dry weight) of various substances soluble in ethanol at 80% (v / v).

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Abstract

The invention concerns dry active yeasts useful for rehydration and/or alcoholic fermentation, or rehydrated active yeasts useful for fermentation, or yeasts useful for fermentation, having a sterol content exhibiting at least two conjugated double bonds at the B cycle of at least 900 νg(m)g/g of dry weight. The invention is useful for producing fermented alcoholic beverages.

Description

« Moyens pour améliorer les capacités fermentaires de levures actives et leurs applications » "Means for improving fermentative capacities of active yeasts and their applications"
L'invention concerne des moyens pour améliorer les capacités fermentaires de levures actives. Elle a plus particulièrement pour objet des levures sèches actives (LSA en abrégé) ou des levures actives réhydratées, à teneur élevée en stérols et leur obtention. L'invention a également pour objet les applications de ces levures dans des processus de fermentation alcoolique, en particulier pour la fabrication de boissons alcooliques fermentées.The invention relates to means for improving the fermentative capacities of active yeasts. It more particularly relates to active dry yeasts (LSA abbreviated) or active yeasts rehydrated, high sterol content and obtaining them. The invention also relates to the applications of these yeasts in alcoholic fermentation processes, in particular for the manufacture of fermented alcoholic beverages.
Les stérols sont des constituants importants de la membrane cellulaire des levures. La concentration en stérols doit être optimale pour assurer l'intégrité membranaire, garantie de l'activité des protéines membranaires, telles que les perméases.Sterols are important constituents of the yeast cell membrane. The concentration of sterols should be optimal to ensure membrane integrity, guaranteeing the activity of membrane proteins, such as permeases.
En conditions fermentaires, si certaines de ces perméases ne fonctionnent plus, on observe une chute de la viabilité cellulaire, pouvant entraîner la mort de la cellule.Under fermentative conditions, if some of these permeases no longer work, there is a drop in cell viability, which can lead to cell death.
La composition en stérols des levures exerce donc un rôle primordial sur- leur capacité fermentaire.The sterol composition of yeasts therefore plays a major role in their fermentative capacity.
Pour augmenter les concentrations en stérols des levures, on a proposé d'incorporer des stérols exogènes pendant la croissance des levures. Mais leur caractère insoluble en milieu aqueux constitue un facteur limitant.To increase yeast sterol concentrations, it has been proposed to incorporate exogenous sterols during yeast growth. But their insoluble nature in an aqueous medium is a limiting factor.
Pour pallier cet inconvénient, on a préconisé l'utilisation d'agents tensio-actifs pour les solubiliser, ce qui limite les possibilités d'addition directe de stérols dans le milieu de culture.To overcome this drawback, it has been advocated the use of surfactants to solubilize, which limits the possibilities of direct addition of sterols in the culture medium.
Dans les cas de carences en facteurs lipidiques, l'ajout de bourbes fines au moût de fermentation a été recommandé afin d'apporter au milieu des nutriments tels que des stérols et des acides gras insaturés sous forme complexe. Un tel ajout n'est toutefois pas toujours facile à réaliser dans des caves.In the case of lipid deficiencies, the addition of fine mashes to the fermentation must has been recommended in order to provide the environment with nutrients such as sterols and complex unsaturated fatty acids. Such an addition is however not always easy to achieve in cellars.
Les inventeurs ont constaté que ces inconvénients pouvaient être surmontés en utilisant des levures inactivées à teneur élevée en certains types de stérols, soit naturellement, soit par enrichissement, ou en utilisant des compositions de stérols sous forme soluble. Il s'est en effet avéré que l'addition de telles levures ou compositions dans le milieu de réhydratation des levures sèches actives ou aux levures actives dans un moût de fermentation, permet de stabiliser les stérols et de disposer de formes solubles capables d'être incorporées efficacement par les levures.The inventors have found that these disadvantages can be overcome by using inactivated yeasts with a high content of certain types of sterols, that is, naturally, either by enrichment, or by using sterol compositions in soluble form. It has indeed been found that the addition of such yeasts or compositions in the rehydration medium of active yeasts or active yeasts in a fermenting wort makes it possible to stabilize the sterols and to have soluble forms capable of being used. incorporated effectively by yeasts.
De manière surprenante, les capacités fermentaires des levures actives du milieu de fermentation sont particulièrement élevées, notamment pendant la phase stationnaire de la fermentation, la durée de la fermentation étant généralement plus courte.Surprisingly, the fermentative capacities of the active yeasts of the fermentation medium are particularly high, especially during the stationary phase of the fermentation, the duration of the fermentation being generally shorter.
L'invention a donc pour objet des levures sèches actives ou des levures actives réhydratées avec des capacités fermentaires améliorées. Elle vise également un procédé de mise en œuvre aisée pour leur obtention.The invention therefore relates to active dry yeasts or rehydrated active yeasts with improved fermentation capacities. It also aims at a method of easy implementation for obtaining them.
L'invention vise en outre la mise à profit des propriétés fermentaires de ces levures actives pour la fabrication de boissons fermentées alcooliques, en particulier de vins, de bières et d'alcools forts comme la vodka ou le whisky.The invention also aims to take advantage of the fermentative properties of these active yeasts for the manufacture of alcoholic fermented beverages, in particular wines, beers and strong alcohols such as vodka or whiskey.
L'invention vise ainsi des LSA utilisables en réhydratation et/ou en fermentation alcoolique, ou des levures actives réhydratées utilisables en fermentation, ou des levures utilisables en fermentation, ayant une teneur en stérols présentant au moins deux doubles liaisons conjuguées au niveau du cycle B d'au moins 200 μg/g de poids sec, et plus spécialement d'au moins 900 μg/g de poids sec.The invention thus provides LSAs that can be used in rehydration and / or alcoholic fermentation, or rehydrated active yeasts that can be used in fermentation, or yeasts that can be used in fermentation, having a sterol content having at least two conjugated double bonds at the B-ring level. at least 200 μg / g dry weight, and more particularly at least 900 μg / g dry weight.
Par « levures utilisables en fermentation », on entend la crème de levures issue de leur production ou les levures congelées.By "yeasts usable in fermentation" means the yeast cream from their production or frozen yeasts.
Il s'agit plus spécialement de levures œnologiques ou de levures de brasserie, ou encore de levures de distillerie, ce qui inclut des levures du genre Saccchromyces ou du genre non Saccchromyces. On citera en particulier des Saccharomyces appartenant aux espèces cerevisiae, uvarum et bayamis.It is more particularly enological yeast or brewer yeasts, or yeasts of distillery, which includes yeasts of the genus Saccchromyces or non Saccchromyces genus. In particular, Saccharomyces belonging to the cerevisiae, uvarum and bayamis species will be mentioned.
L'enrichissement de LSA en stérols est obtenu avantageusement par incorporation de compositions de stérols sous forme soluble à partir de micelles. Avantageusement, ces micelles comprennent au moins 10 à 20 mg/g (poids sec) de stérols, en particulier au moins 15 mg/g (poids sec), et environ 600 à 800 mg/g (poids sec) de complexes lipopolysaccharides - protéines, précipitables par Péthanol à 80% (v/v), en particulier 660 à 670 mg/g (poids sec), d'un poids moléculaire moyen de 160 àThe enrichment of LSA with sterols is advantageously obtained by incorporation of sterol compositions in soluble form from micelles. Advantageously, these micelles comprise at least 10 to 20 mg / g (dry weight) of sterols, in particular at least 15 mg / g (dry weight), and approximately 600 to 800 mg / g (dry weight) of lipopolysaccharide-protein complexes. , precipitated with 80% (v / v) ethanol, in particular 660 to 670 mg / g (dry weight), with an average molecular weight of 160 to
17O kDa environ.17O kDa approx.
Les stérols comprennent au moins 20 - 25% d'ergostérol et 20 - 25% de zymostérol, d'autres stérols étant présents, comme les fécostérol, lanostérol ou ergosta 5 - 7 diénols, sans que cette énumération soit limitative.The sterols comprise at least 20-25% ergosterol and 20-25% zymosterol, other sterols being present, such as ergosterol, lanosterol or ergosta 5 - 7 dienols, without this enumeration being limiting.
Une composition de telles micelles est donnée dans les exemples. Selon un mode de réalisation de l'invention, les LSA enrichies en stérols sont obtenues par addition dans leur milieu de réhydratation ou dans le moût de fermentation de levures inactivées, ayant elles-mêmes une teneur en stérols présentant au moins deux doubles liaisons conjuguées au niveau du cycle B d'au moins 200 μg/g de poids sec, et plus spécialement d'au moins 900 μg/g ( poids sec).A composition of such micelles is given in the examples. According to one embodiment of the invention, the sterol-enriched LSAs are obtained by addition in their rehydration medium or in the fermentation mash of inactivated yeasts, themselves having a sterol content having at least two conjugated double bonds at the same time. B cycle level of at least 200 μg / g dry weight, and more particularly at least 900 μg / g (dry weight).
De telles levures inactivées sont des levures naturellement riches en stérols ou des levures enrichies en stérols, notamment en ergostérol, et/ou en zymostérol, et/ou ergosta 5-7 diénols, avantageusement selon les techniques connues de l'homme du métier.Such inactivated yeasts are yeasts naturally rich in sterols or yeasts enriched in sterols, in particular ergosterol, and / or zymosterol, and / or ergosta 5-7 dienols, advantageously according to the techniques known to those skilled in the art.
De préférence, les levures utilisées ont avantageusement une teneur en stérols possédant deux doubles liaisons conjuguées au niveau du cycle B, de 900 à 1 250 μg/g de poids sec.Preferably, the yeasts used advantageously have a sterol content having two conjugated double bonds in ring B, from 900 to 1250 μg / g dry weight.
Les quantités de levures inactivées ajoutées dans le milieu de réhydratation sont de l'ordre de 20 à 500g/l, de préférence de 100 à 200 g/1, notamment de l'ordre de 150g/l.The amounts of inactivated yeasts added to the rehydration medium are of the order of 20 to 500 g / l, preferably 100 to 200 g / l, in particular of the order of 150 g / l.
L'enrichissement des LSA s'effectue grâce au transfert des stérols sous forme de micelles à partir des levures inactivées.LSA is enriched by transferring sterols in the form of micelles from inactivated yeasts.
Selon un autre mode de réalisation de l'invention, l' enrichissement des levures en stérols est obtenu par addition, à des LSA dans leur milieu de réhydratation ou à des LSA ajoutées à des moûts de fermentation, d'une composition lipidique renfermant une fraction de stérols comprenant de Tergostérol et du zymostérol et un complexe de lipopolysaccharides — protéines précipitables par Péthanol .According to another embodiment of the invention, the yeast sterol enrichment is obtained by addition to LSA in their rehydration medium or to LSA. added to fermentation mashes, a lipid composition containing a sterol fraction comprising ergosterol and zymosterol and a complex of lipopolysaccharides - proteins precipitable with ethanol.
Ces compositions renferment avantageusement de 10 à 70 % en poids sec d'ergostérol et de 10 à 70% en poids sec de zymostérol, à raison de 10 à 20 mg de stérols totaux par g. et de 500 à 1000 mg/g de complexe lipopolysaccharides-protéines.These compositions advantageously contain from 10 to 70% by dry weight of ergosterol and from 10 to 70% by dry weight of zymosterol, in a proportion of 10 to 20 mg of total sterols per g. and from 500 to 1000 mg / g of lipopolysaccharide-protein complex.
Avantageusement, la fraction stérolique comprend en outre un ou plusieurs stérols tels que le fécostérol, le lanostérol, les ergosta 5 — 7 diénols (dihydroergostérol, méthylzymostérol, desméthyllanostérol, et ergosta-7,22-dièn-3-ol).Advantageously, the sterol fraction further comprises one or more sterols such as fecosterol, lanosterol, ergosta 5-7 dienols (dihydroergosterol, methylzymosterol, desmethyllanosterol, and ergosta-7,22-dien-3-ol).
Les compositions lipidiques telles qu'utilisées dans le procédé ci-dessus constituent des produits nouveaux et, à ce titre, entrent également dans le champ de l'invention.The lipid compositions as used in the above process are novel products and, as such, are also within the scope of the invention.
Ces compositions lipidiques possèdent notamment les propriétés physico-chimiques suivantes :These lipid compositions have in particular the following physicochemical properties:
- elles correspondent à une forme colloïdale amphiphile sans solubilité vraie en suspension dans de l'eau pure à 40C, - elles ne peuvent pas traverser des membranes de filtrations d'un seuil de coupure de 0.2μm, elles présentent un effet tensio-actif qui ne se manifeste qu'au-dessus d'une concentration de 0.5g/l en poids sec en phase aqueuse,- they correspond to an amphiphilic colloidal form without true solubility suspended in pure water at 4 0 C, - they can not pass through the filtration membranes of a 0.2μm cut-off threshold, they have an effect surfactant active which is manifested only above a concentration of 0.5g / l in dry weight in aqueous phase,
- elles ont la propriété de s'auto agréger au-dessus d'une concentration de 0.05 g/1 en poids sec en phase aqueuse pour former des agrégats micellaires d'un diamètre moyen d'environ 380 nm (valeur obtenue par mesure de la diffraction de lumière laser) par technique PCS (Photo Corrélation Spetroscopy).they have the property of self-aggregating above a concentration of 0.05 g / l by dry weight in aqueous phase to form micellar aggregates with an average diameter of approximately 380 nm (value obtained by measuring the diffraction of laser light) by PCS technique (Photo Correlation Spetroscopy).
Pour obtenir un effet tensio-actif maximal des stérols, les compositions ci-dessus sont utilisées à raison de 1,5 à 3 g/1, de préférence de 2g/l de milieu de réhydratation ou de 20 à 1 OOg/hecto de moût de fermentation.To obtain a maximum surfactant effect of the sterols, the above compositions are used in a proportion of 1.5 to 3 g / l, preferably 2 g / l of rehydration medium or 20 to 100 g / hectare of must. fermentation.
Les levures à teneur élevée en stérols, telles qu'obtenues par enrichissement pendant leur phase de réhydratation ou de fermentation, grâce au transfert et à l'incorporation des stérols sous forme de micelles à partir des levures inactivées, ou par incorporation desdites compositions lipidiques, présentent un grand intérêt dans les processus fermentaires compte tenu de leurs capacités fermentaires élevées durant la phase stationnaire des fermentations.Yeasts with a high sterol content, as obtained by enrichment during their rehydration or fermentation phase, through the transfer and incorporation of Sterols in the form of micelles from inactivated yeasts, or by incorporation of said lipid compositions, are of great interest in fermentative processes in view of their high fermentation capacities during the stationary phase of fermentations.
En outre, cette teneur élevée en stérols diminue la production d'acides volatiles, ce qui permet d'améliorer les propriétés organoleptiques des produits de fermentation.In addition, this high sterol content decreases the production of volatile acids, which improves the organoleptic properties of the fermentation products.
L'invention vise ainsi également l'utilisation, comme auxiliaires technologiques, de levures inactivées riches en stérols, comme défini ci-dessus, dans des milieux de réhydratation de LSA ou dans des milieux de fermentation alcoolique, pour la production de boissons fermentées alcooliques, en particulier de vins ou encore de bières, ou d'alcools de type whisky, vodka et autres.The invention thus also relates to the use, as processing aids, of inactivated yeasts rich in sterols, as defined above, in LSA rehydration media or in alcoholic fermentation media, for the production of alcoholic fermented beverages, especially wines or beers, or alcohols whiskey, vodka and others.
. L'invention vise également un procédé de production de telles boissons comportant l'addition de levures sèches actives enrichies en stérols comme défini ci-dessus, à raison de. The invention also relates to a process for producing such beverages comprising the addition of sterol-enriched active dry yeasts as defined above, on the basis of
100 à 200 g/1 de milieu de fermentation, de préférence 150g/l. Ces levures sont soit des levures naturellement riches en stérols, réhydratées ou non, soit des levures enrichies comme défini ci-dessus, à savoir des levures actives réhydratées ou des LSA.100 to 200 g / l of fermentation medium, preferably 150 g / l. These yeasts are either yeasts naturally rich in sterols, rehydrated or not, or yeasts enriched as defined above, namely rehydrated active yeasts or LSA.
D'autres caractéristiques et avantages de l'invention sont donnés dans les exemples qui suivent. Dans ces exemples, il sera fait référence aux figues 1 et 2, qui représentent, respectivement,Other features and advantages of the invention are given in the examples which follow. In these examples, reference will be made to figs 1 and 2, which represent, respectively,
- la figure 1 la réhydratation d'une LSA en présence de préparations de stérols selon l'invention, à teneurs différents en stérols ; et - la figure 2, l'incorporation de stérols par des LSA pendant une phase de réhydratation en utilisant une préparation de stérols selon l'invention.FIG. 1 rehydration of an LSA in the presence of sterol preparations according to the invention, with different contents of sterols; and - Figure 2, the incorporation of sterols by LSA during a rehydration phase using a sterol preparation according to the invention.
Exemple 1 : Formulation de stérols stabilisés sous forme solubleExample 1: Formulation of Stabilized Sterols in Soluble Form
On soumet 10 ml de suspension dans l'eau d'un échantillon de levures inactivées, à une concentration de 150 g/1 de poids sec, à une étape d'incubation, sous agitation, à 37°C, pendant 30 min.10 ml of suspension in water of an inactivated yeast sample, at a concentration of 150 g / l of dry weight, is subjected to an incubation stage, with stirring, at 37 ° C. for 30 min.
Par centrifugation de la préparation à 11 300g, 15 min., on obtient un surnageant 1 et un culot L Le surnageant i est soumis à une étape d'ultracentrifugation à 85 000 g, 3h, à 4°C. On obtient un culot 2 un surnageant 2 et un colloïde blanc en surface. La fraction colloïdale blanche est lyophilisée pour donner la préparation de stérols stabilisés sous forme soluble (environ 20 mg).By centrifugation of the preparation at 11,300g for 15 min, a supernatant 1 and a pellet L are obtained. The supernatant is subjected to an ultracentrifugation step at 85,000 g, 3h, at 4 ° C. A pellet 2, a supernatant 2 and a white colloid are obtained on the surface. The colloidal fraction white is lyophilized to give the stabilized sterol preparation in soluble form (about 20 mg).
Exemple 2 : Effet de la formulation de stérols selon l'exemple 1 sur le profil de fermentation d'un moût synthétiqueEXAMPLE 2 Effect of the Sterol Formulation According to Example 1 on the Fermentation Profile of a Synthetic Mash
Ig de levure sèche active LALVIN ECl 118 ® est réhydraté dans 10ml d'un milieu de réhydratation constitué par l'eau additionnée de 50g/l de glucose. Les réhydratations sontActive dry yeast Ig LALVIN ECl 118 ® is rehydrated in 10ml of a rehydration medium consisting of water with 50g / l of glucose. Rehydration is
• effectuées à 37°C pendant 30 min, en présence de 12 ou de 24mg de la formulation de stérols de l'exemple 1, et à titre de contrôle, en l'absence de formulation de stérol. Deux fermentations ont été conduites en parallèle et en duplicata sur un moût synthétique MS300-stigmastérol correspondant à un moût riche en azote (300 mg d' azote/1), carence en stérols et contenant 200g/l de sucres fermentescibles. • carried out at 37 ° C for 30 min in the presence of 12 or 24mg of the sterol formulation of Example 1, and as a control, in the absence of sterol formulation. Two fermentations were conducted in parallel and in duplicate on an MS300-stigmasterol synthetic must corresponding to a must rich in nitrogen (300 mg of nitrogen / 1), sterol deficiency and containing 200g / l of fermentable sugars.
Quatre fermenteurs contenant 1.11 de moût synthétique MS300-stigmastérol sont inoculés avec 2.2ml de solution de réhydratation. La température est maintenue à 240C. La production de CO2 est relevée en fonction du temps.Four fermentors containing 1.11 of MS300-stigmasterol synthetic must are inoculated with 2.2ml of rehydration solution. The temperature is maintained at 24 ° C. The production of CO 2 is recorded as a function of time.
Les résultats obtenus sont donnés sur la figure 1. L'examen de cette figure montre que les fermentations conduites par les levures réhydratées en présence de formulation de stérols utilisée se terminent avant les fermentations témoins.The results obtained are given in FIG. 1. The examination of this figure shows that the fermentations conducted by the rehydrated yeasts in the presence of the sterol formulation used end before the control fermentations.
Globalement, les cinétiques observées pour les fermentations conduites par les levures réhydratées en présence de la formulation de stérols stabilisés sous forme soluble sont plus . rapides dès l'entrée en phase stationnaire.Overall, the kinetics observed for the fermentations conducted by the rehydrated yeasts in the presence of the formulation of sterols stabilized in soluble form are more. fast when entering the stationary phase.
Exemple 3 : Effet de la- formulation de stérols selon l'exemple 1 sur l'activation de l'incorporation de stérols par les LSA On opère comme dans l'exemple 1, mais on effectue la réhydratation en présence de 2 μl de [4-14C] cholestérol (50-60 mCi/mMol (Amersham CFA 128)), solubilisé dans 20 mμl de mélange tergitol/éthanol (v/v) en présence ou non de 24mg de la formulation de stérols dé l'exemple 1 pour la fabrication de boissons fermentées alcooliques.EXAMPLE 3 Effect of Formulation of Sterols According to Example 1 on the Activation of the Incorporation of Sterols by LSAs The procedure is as in Example 1, but the rehydration is carried out in the presence of 2 μl of [4] - 14 C] cholesterol (50-60 mCi / mMol (Amersham CFA 128)), solubilized in 20 mμl of tergitol / ethanol mixture (v / v) in the presence or absence of 24 mg of the sterol formulation of Example 1 for the manufacture of alcoholic fermented beverages.
On mesure la radioactivité incorporée au cours du temps par les LSA en procédant comme suit :The radioactivity incorporated over time by the LSAs is measured as follows:
On filtre 300 μl de milieu sous vide partiel sur filtre en fibres de verre Whatman GF/C, on rince par 3ml d'eau distillée à 40C, on sèche et compte par scintillation liquide sur compteur Beckman LS 6500, après ajout de 20ml de liquide scintillant Fluoransafe 2® (BDH). Les résultats obtenus sont rapportés sur la figure 2. On constate que pendant la réhydratation, la présence de la formulation de stérols selon l'invention permet une incorporation plus efficace des stérols du milieu par les LSA.300 μl of medium under partial vacuum are filtered through a Whatman GF / C glass fiber filter, rinsed with 3 ml of distilled water at 40 ° C., dried and counted by liquid scintillation on a Beckman LS 6500 counter, after the addition of 20 ml. Fluoransafe 2® (BDH). The results obtained are reported in FIG. 2. It is noted that during rehydration, the presence of the sterol formulation according to the invention allows a more efficient incorporation of the sterols of the medium by the LSAs.
Exemple 4 : Enrichissement en stérols de levures inactivéesExample 4 Enrichment of inactivated yeasts with sterols
Les souches de levure sont cultivées sous une aération constante et forte. La température des propagations est de l'ordre de 15 à 40°C, de préférence de 25 à 35°C.The yeast strains are grown under constant and strong aeration. The temperature of the propagation is of the order of 15 to 40 ° C, preferably 25 to 35 ° C.
La culture des levures est effectuée par apport constant de sources de carbone et de sources d'azote, ainsi que par un apport intermittent en facteurs de croissance essentiels. Les sources de carbone dérivent généralement de molasses et peuvent être des molasses de sucre, de betterave ou leur mélange à des concentrations variables. La source d'azote peut provenir de divers dérivés ammoniaque, comme l'hydroxyde d'ammoniaque, le chlorure d'ammonium, le sulfate d'ammonium, le phosphate d'ammonium, le phosphate de di-ammonium ou d'extraits protéiques bruts. La propagation de la levure est réalisée selon un programme comportant des variations des vitesses de croissance. Les vitesses de croissance maximales sont de 0.05/h à 0.25/h, de préférence de 0.15 à 0.2/h.Yeast culture is carried out by constant supply of carbon sources and nitrogen sources, as well as intermittent input of essential growth factors. The carbon sources are generally derived from molasses and may be molasses of sugar, beet or their mixture in varying concentrations. The nitrogen source can come from various ammonia derivatives, such as ammonia hydroxide, ammonium chloride, ammonium sulfate, ammonium phosphate, di-ammonium phosphate or crude protein extracts . The propagation of the yeast is carried out according to a program comprising variations in the growth rates. The maximum growth rates are from 0.05 / h to 0.25 / h, preferably from 0.15 to 0.2 / h.
L'enrichissement de la levure en stérols peut être effectué en modifiant de manière spécifique les sources de carbone et d'azote afin de réduire le taux total d'azote par rapport à celui du carbone.Enrichment of yeast sterols can be done by specifically modifying the carbon and nitrogen sources to reduce the total nitrogen rate relative to that of carbon.
Il est également possible d'ajouter des précurseurs de stérols spécifiques de manière contrôlée. Des sources de précurseurs de stérols préférées comprennent l'éthanol, l'acide acétique, le squalène ou le lanostérol. Ces précurseurs peuvent être ajoutés sous forme purifiée ou brute. - .It is also possible to add specific sterol precursors in a controlled manner. Preferred sterol precursor sources include ethanol, acetic acid, squalene or lanosterol. These precursors may be added in purified or crude form. -.
Exemple 5 : Compositions lipidiques utilisées comme additifs dans des milieux de réhydratation de levures et/ou dans des moûts de fermentationEXAMPLE 5 Lipid Compositions Used as Additives in Yeast Rehydration Media and / or in Ferment Mashes
Ces compositions sont obtenues par un procédé comprenant les étapes : - d'incubation sous agitation d'une suspension dans l'eau de levures œnologiques inactivées dans des conditions permettant l'obtention de micelles formées de fractions spécifiquement enrichies en stérols incorporés dans des complexes de lipides - protéines,These compositions are obtained by a process comprising the following steps: incubation with stirring of a suspension in water of inactivated oenological yeasts under conditions making it possible to obtain micelles formed of fractions specifically enriched with sterols incorporated into complexes of lipids - proteins,
- de séparation des fractions micellaires, suivie de lyophilisation. L'étape de séparation, après l'incubation, des fractions micellaires est plus spécialement réalisée par centrifugation. Le surnageant recueilli est soumis alors à une étape d'ultracentrifugation pour augmenter l'enrichissement de la fraction en micelles de stérols, la fraction colloïdale qui surnage est isolée et lyophilisée.separation of the micellar fractions, followed by freeze-drying. The separation step, after the incubation, of the micellar fractions is more particularly carried out by centrifugation. The collected supernatant is then subjected to an ultracentrifugation step to increase the enrichment of the sterol micelle fraction, the supernatant colloid fraction is isolated and lyophilized.
Les compositions obtenues renferment une fraction de stérols composés de 10 à 70 %, notamment de 20 à 25% en poids sec d'ergostérol, de 10 à 70 %, notamment de 20 à 25 % en poids sec de zymostérol à raison de 10 à 20 mg de stérols totaux par g, et un complexe de lipopolysaccharides-protéines précipitables par Péthanol à 80% (v/v), d'un poids moléculaire moyen de l'ordre de 160-170 kDa, à raison de 600 à 800 mg/g.The compositions obtained contain a fraction of sterols composed of 10 to 70%, especially 20 to 25% by dry weight of ergosterol, of 10 to 70%, especially 20 to 25% by dry weight of zymosterol in a proportion of 10 to 20 mg of total sterols per g, and a complex of 80% (v / v) ethanol-precipitable lipopolysaccharide proteins, with an average molecular weight of about 160-170 kDa, at 600 to 800 mg /boy Wut.
La fraction stérolique comprend en outre un ou plusieurs stérols, tels que le fécostérol, le lanostérol, et les ergostastérols 5-7 diénols.The sterol fraction further comprises one or more sterols, such as fecosterol, lanosterol, and ergostasterols 5-7 dienols.
Dans le complexe de lipolysaccharides - protéines, les polysaccharides, les lipides et les protéines sont avantageusement présents à raison deIn the lipolysaccharide-protein complex, the polysaccharides, the lipids and the proteins are advantageously present on the basis of
. 20 à 34%, notamment de 27% pour les polysaccharides, . 55 à 70% notamment 61% pour les lipides, et . 10 à 15%, notamment 12% pour les protéines.. 20 to 34%, especially 27% for polysaccharides,. 55 to 70% in particular 61% for lipids, and. 10 to 15%, especially 12% for proteins.
Des polysaccharides préférés ont un PM moyen de l'ordre de 30-35 kDa, en particulier de 30 à 32 kDa et contiennent du mannose du glucose et de l'inositol. Des proportions respectives appropriées correspondent àPreferred polysaccharides have an average MW of about 30-35 kDa, especially 30-32 kDa, and contain mannose of glucose and inositol. Appropriate proportions respectively correspond to
. 55 à 70%, notamment 61% pour le mannose, . 30 à 40%, notamment 33% pour le glucose, . 0,5 à 1%. notamment 0,9% pour l'inositol.. 55 to 70%, especially 61% for mannose,. 30 to 40%, especially 33% for glucose, 0.5 to 1%. especially 0.9% for inositol.
La fraction lipidique comprend, en quantités variables, de préférence des acides gras Cl 7 :1, C16 :0 et Cl 8 :0. Cette fraction lipidique est liée aux polysaccharides par l'intermédiaire d'un groupe phosphatidylinositol.The lipid fraction comprises, in varying amounts, preferably Cl 7: 1, C 16: 0 and Cl 8: 0 fatty acids. This lipid fraction is bound to the polysaccharides via a phosphatidylinositol group.
Le restant de la préparation est formé de fragments membranaires solubles dans l'éthanol ) 80% (v/v).The remainder of the preparation is 80% (v / v) ethanol soluble membrane fragments.
Les micelles formées sont par exemple composés de : 1) Au moins 15 mg/g (poid sec) de stérols, composés d'au moins 20-25% d'ergostérol, 20-25% de zymostérol et des quantités variables d'autres stérols (fécostérol, lanostérol, ergosta 5-7 diénols, etc.). 2) 667 mg/g (poids sec) de complexes lipopolysaccharides-protéines, précipitables par l'éthanol à 80% (v/v), d'un poids moléculaire moyen de 167 kDa, composés de 27% de polysaccharides, 61% de lipides en poids et 12% de protéines. Les polysaccharides ont un poids moléculaire moyen de 31,7 kDa, et contiennent 61% de mannose, 33% de glucose et 0,9% d'inositol. La fraction lipidique est formée des acides gras C17 :1, C16 :0 et C18 :0 en quantités variables. La fraction lipidique est liée aux polysaccharides par le biais d'une ancre phosphatidyl inositol. 3) 333 mg/g (poids sec) de substances diverses solubles dans l'éthanol à 80% (v/v). The micelles formed are for example composed of: 1) At least 15 mg / g (dry weight) of sterols, composed of at least 20-25% of ergosterol, 20-25% of zymosterol and varying amounts of other sterols (fecosterol, lanosterol, ergosta 5- 7 dienols, etc.). 2) 667 mg / g (dry weight) of 80% (v / v) ethanol-precipitable lipopolysaccharide-protein complexes, with an average molecular weight of 167 kDa, composed of 27% of polysaccharides, 61% of lipids by weight and 12% of proteins. The polysaccharides have an average molecular weight of 31.7 kDa, and contain 61% mannose, 33% glucose and 0.9% inositol. The lipid fraction is formed of C17: 1, C16: 0 and C18: 0 fatty acids in varying amounts. The lipid fraction is bound to the polysaccharides via a phosphatidyl inositol anchor. 3) 333 mg / g (dry weight) of various substances soluble in ethanol at 80% (v / v).

Claims

REVENDICATIONS
1/ Levures sèches actives utilisables en réhydratation et/ou en fermentation alcoolique, ou des levures actives réhydratées utilisables en fermentation, ou des levures utilisables en fermentation, ayant une teneur en stérols présentant au moins deux doubles liaisons conjuguées au niveau du cycle B d'au moins 200 μg/g de poids sec.1 / Active dry yeasts that can be used in rehydration and / or alcoholic fermentation, or rehydrated active yeasts that can be used in fermentation, or yeasts that can be used in fermentation, having a sterol content having at least two conjugated double bonds at the B-ring level at least 200 μg / g dry weight.
2/ Levures sèches actives selon la revendication 1, caractérisées en ce qu'il s'agit de levures œnologiques ou de levures de brasserie ou encore de levures de distillerie, incluant des levures du genre Saccharomyces ou du genre non Saccharomyces.2 / active dry yeasts according to claim 1, characterized in that it is oenological yeasts or brewer yeast or distillery yeasts, including yeasts of the genus Saccharomyces or non-Saccharomyces genus.
3/ Levures. sèches actives selon la revendication 2 caractérisées en ce qu'elles appartiennent au genre Sacchoromyces, en particulier des espèces cerevisiae, uvarum et bayanus.3 / Yeasts. dry agents according to claim 2 characterized in that they belong to the genus Sacchoromyces, in particular species cerevisiae, uvarum and bayanus.
4/ Procédé d'obtention de levures actives selon l'une quelconque des revendications 1 à 3, caractérisées par l'enrichissement en . stérols des levures sèches actives par incorpo de compositions de stérols sous forme soluble à partir de micelles.4 / Process for obtaining active yeasts according to any one of claims 1 to 3, characterized by the enrichment. sterols of active dry yeasts by incorporating sterol compositions in soluble form from micelles.
5/ Procédé selon la revendication 4, caractérisé en ce que les LSA enrichies en stérols sont obtenues par ration addition dans leur milieu de réhydratation ou dans le moût de fermentation de levures inactivées, ayant elles-mêmes une teneur en stérols présentant au moins deux doubles liaisons conjuguées au niveau du cycle B d'au moins 200 μg/g de poids sec, et plus spécialement d'au moins 900 μg/g (poids sec).5 / A method according to claim 4, characterized in that the sterol-enriched LSAs are obtained by ration addition in their rehydration medium or in the fermentation must of inactivated yeasts, themselves having a sterol content having at least two double conjugate bonds at the B-ring level of at least 200 μg / g dry weight, and more especially at least 900 μg / g (dry weight).
6/ Procédé selon la revendication 4 ou 5, caractérisé en ce que lesdites levures inactivées sont des levures naturellement riches en stérols ou des levures enrichies en stérols, notamment en ergostérol, et/ou en zymostérol, et/ou ergosta 5-7 diénols.6 / A method according to claim 4 or 5, characterized in that said inactivated yeasts are yeasts naturally rich in sterols or yeasts enriched in sterols, especially ergosterol, and / or zymosterol, and / or ergosta 5-7 dienols.
11 Procédé selon la revendication 5 ou 6, caractérisé en ce que lesdites levures ont une teneur en stérols possédant deux doubles liaisons conjuguées au niveau du cycle B, de 900 à 1 250 μg/g (poids sec). 8/ Procédé selon l'une quelconque des revendications 5 à 7, caractérisé en ce que les quantités de levures inactivées ajoutées dans le milieu de réhydratation sont de l'ordre de 20 à 500g/ll.11. The method of claim 5 or 6, characterized in that said yeasts have a sterol content having two conjugated double bonds in the B cycle, 900 to 1250 μg / g (dry weight). 8 / A method according to any one of claims 5 to 7, characterized in that the amounts of inactivated yeasts added to the rehydration medium are of the order of 20 to 500g / ll.
9/ Procédé selon la revendication 4, caractérisé en ce que l'enrichissement des levures sèches actives en stérols est réalisé par addition, dans leur milieu de réhydratation, ou à des LSA de moûts de fermentation, d'une composition lipidique renfermant :9 / A method according to claim 4, characterized in that the enrichment of sterol-active dry yeasts is carried out by adding, in their rehydration medium, or to LSA of fermentation musts, a lipid composition containing:
. une fraction de stérols comprenant de l'ergostérol et du zymostérol et. a sterol fraction comprising ergosterol and zymosterol and
. un complexe de lipopolysaccharides - protéines précipitables par l'éthanol.. a complex of lipopolysaccharides - proteins precipitable by ethanol.
10/ Procédé selon la revendication 9, caractérisé en ce que les compositions lipidiques renferment de 10 à 70 % en poids sec d'ergostérol et de 10 à 70% en poids sec de zymostérol, à raison de 10 à 20 mg de stérols totaux par g. et de 500 à 1000 mg/g de complexe lipopolysaccharides-protéines .10 / A method according to claim 9, characterized in that the lipid compositions contain from 10 to 70% by dry weight of ergosterol and 10 to 70% by dry weight of zymosterol, at a rate of 10 to 20 mg of total sterols by boy Wut. and from 500 to 1000 mg / g of lipopolysaccharide-protein complex.
11/ Procédé selon la revendication 9 ou 10, caractérisé en ce que la fraction stérolique comprend en outre un ou plusieurs stérols tels que le fécostérol, le lanostérol, les ergosta - 5 - 7 diénols.11 / A method according to claim 9 or 10, characterized in that the sterol fraction further comprises one or more sterols such as fecterol, lanosterol, ergosta-5-dienols.
12/ Procédé selon l'une quelconque des revendications 9 à 11, caractérisé par l'addition desdites compositions, à raison de 1,5 à 3 g/1, de préférence de 2g/l de milieu de réhydratation ou de 20à lOOg/hl de moût de fermentation12 / A method according to any one of claims 9 to 11, characterized by the addition of said compositions, at a rate of 1.5 to 3 g / 1, preferably 2 g / l of rehydration medium or 20 to 100 g / hl of fermentation must
13/ Utilisation, comme auxiliaires technologiques, de levures inactivées riche en stérols, telles que mises en œuvre dans le procédé de l'une quelconque des revendications 5 à 8, dans des milieux de réhydratation de LSA ou dans des milieux de fermentation alcoolique, aux fins de production d'une boisson par fermentation alcoolique.13 / Use, as processing aids, inactivated yeasts rich in sterols, as implemented in the process of any one of claims 5 to 8, in LSA rehydration media or in alcoholic fermentation media, to purposes of producing a drink by alcoholic fermentation.
14/ Procédé de production de boissons fermentées alcooliques, caractérisé en ce qu'il comprend l'utilisation d'au moins une levure sèche active ayant une teneur en stérols, ou d'une levure active réhydratée, selon l'une quelconque des revendications 1 à 3, ou telle qu'obtenue par le procédé selon l'une quelconque des revendications 4 à 12.14 / A process for producing alcoholic fermented beverages, characterized in that it comprises the use of at least one active dry yeast having a sterol content, or a rehydrated active yeast, according to any one of claims 1 at 3, or as obtained by the method according to any one of claims 4 to 12.
15/ Procédé selon la revendication 14, caractérisé en ce qu'il comprend l'addition de la ou desdites levures à raison de 100 à 200 g/1 de milieu de fermentation, de préférence 150 g/1. 16/ Application du procédé selon l'une quelconque des revendications 14 ou 15 à la production de vins, de bières, ou d'alcools de type whisky, vodka ou autres. 15 / A method according to claim 14, characterized in that it comprises the addition of said yeast (s) at a rate of 100 to 200 g / l of fermentation medium, preferably 150 g / l. 16 / Application of the method according to any one of claims 14 or 15 for the production of wines, beers, or alcohols of the whiskey, vodka or other type.
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