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WO2017114999A1 - Rabotures avec des microorganismes, leur préparation et leur utilisation - Google Patents

Rabotures avec des microorganismes, leur préparation et leur utilisation Download PDF

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Publication number
WO2017114999A1
WO2017114999A1 PCT/ES2016/070951 ES2016070951W WO2017114999A1 WO 2017114999 A1 WO2017114999 A1 WO 2017114999A1 ES 2016070951 W ES2016070951 W ES 2016070951W WO 2017114999 A1 WO2017114999 A1 WO 2017114999A1
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WIPO (PCT)
Prior art keywords
chips
microorganisms
oak
yeasts
immobilized
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PCT/ES2016/070951
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English (en)
Spanish (es)
Inventor
Sergio FERRER SOLER
Isabel PARDO CUBILLOS
Carmen BERBEGAL DE GRACIA
Olga LUCIO COSTA
Lucía POLO TARÍN
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Universitat De València
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Publication of WO2017114999A1 publication Critical patent/WO2017114999A1/fr

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    • 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
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
    • C12G1/00—Preparation of wine or sparkling wine
    • C12G1/02—Preparation of must from grapes; Must treatment and fermentation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
    • C12C11/00—Fermentation processes for beer
    • C12C11/09—Fermentation with immobilised yeast
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
    • C12G1/00—Preparation of wine or sparkling wine
    • C12G1/02—Preparation of must from grapes; Must treatment and fermentation
    • C12G1/0203—Preparation of must from grapes; Must treatment and fermentation by microbiological or enzymatic treatment

Definitions

  • the present invention relates to oak chips (chips) in which microorganisms have been immobilized. More specifically, the invention relates to oak shavings in which microorganisms involved in winemaking have been immobilized, by lyophilization, especially yeasts capable of initiating or carrying out alcoholic fermentation, which may be co-immobilized with bacteria that They carry out malolactic fermentation.
  • the use of said oak chips is also part of the invention, as well as the process of obtaining them.
  • the wine is defined as the alcoholic beverage resulting from the fermentation, total or partial, of the fresh grape, crushed or not, or its must.
  • Sparkling wine is the product obtained through the first or second alcoholic fermentation of, among others, fresh grapes, grape must, wine (all of them suitable for obtaining table wine) or table wine which, when the container is opened, carbon dioxide derived exclusively from fermentation and kept at the temperature of 20 C in closed containers and has an excess pressure due to carbon dioxide in less than 3 bars.
  • sparkling wines are obtained by two fermentations, one of the grape must that is transformed into the base wine in open tanks; In this first fermentation the sugars of the must are transformed into ethanol and C0 2, which escapes into the air. Then this base wine performs a second fermentation in closed containers.
  • the second fermentation of sparkling wines can take place in bottles or in large sealed containers, from which it is transferred to bottles for commercialization.
  • the latter are called sparkling "fermentation in closed vat", “large containers” or “granvas”.
  • Sparkling wines made by the traditional method originating in the French region of Champagne are highly valued, in which C0 2 is obtained through a second alcoholic fermentation in the bottle, induced by the addition of yeasts (usually , of the species Saccharomyces cerevisiae) and, usually, additional sugar.
  • yeasts usually , of the species Saccharomyces cerevisiae
  • additional sugar usually, additional sugar.
  • the bottle remains in a horizontal position for at least 9 months; then it tilts with the neck down and, in this position, it becomes rotate the bottle to favor dead yeasts and other sediments or grounds being deposited in the lower part of the neck.
  • These sediments will be removed by freezing the portion of the sparkling wine that contains them and opening the cap, which is triggered by the effect of pressure, dragging the frozen sediment.
  • the cap is replaced by another one before its commercialization.
  • the winemaking process includes two stages carried out by microorganisms, alcoholic fermentation (FA) and malolactic fermentation (FML).
  • FA alcoholic fermentation
  • FML malolactic fermentation
  • FA is essential.
  • yeasts mainly Saccharomyces cerevisiae, although it is not the only one involved in the process.
  • yeasts mainly Saccharomyces cerevisiae
  • non-Saccharomyces that participate in AF, especially in the early stages giving greater aromatic complexity to the wine.
  • Hanseniospora Hanseniospora, Kloeckera, Torulospora or Metschnikovia (Ribéreau-Gayon et al. 2000).
  • Spontaneous fermentation is of great importance, because endemic yeasts provide organoleptic characteristics typical of the area and wines that are considered more "natural” are obtained; however, the quality of the product can be very variable (Escalante et al., 2007). Therefore, in recent times, this spontaneous fermentation has been passed to the inoculation of strains of oenological interest, selected based on technological criteria and quality of the final product, such as good fermentation capacity or the production of adequate organoleptic characteristics . This selection has been made possible by the studies that have been carried out on the different species of yeasts involved in fermentation, the phases of the process in which they appear and the aromatic compounds to which they give rise.
  • Saccharomyces cerevisiae has been considered the most important yeast species in the wine production process.
  • Saccharomyces cerevisiae it is common to refer to the yeasts present during winemaking by classifying them into two groups: Saccharomyces cerevisiae and non-Saccharomyces yeasts.
  • non-Saccharomyces yeasts isolated from musts in the initial moments of winemaking are also Krutators, their activity is less than that of S. cerevisiae, so it quickly surpasses them in number.
  • non-Saccharomyces yeasts are considered important to define the aromatic profile of the wines, because these yeasts give rise to enzymatic reactions that give rise to a wide range of volatile and non-volatile end products (Romancino et al., 2008 ). Although most die during the initial stages of fermentation because the increase in alcohol concentration is toxic to them, it has been shown that some survive during fermentation and that the metabolites generated by some non-Saccharomyces species can contribute to quality Of the wine.
  • the FML for its part, is essential for the quality of wine, especially in red wine.
  • This fermentation is carried out by lactic bacteria (BAL), mainly by Oenococcus oeni, and in it the L-malic acid present in the wine is transformed into L-lactic acid freeing C0 2 (Kunkee 1991 ) .
  • Lactic acid contains only one acidic group, instead of two of the malic acid, the FML therefore, reduces the total acidity of the wine and increases its pH.
  • the taste of malic acid is more unpleasant (herbaceous, aggressive taste) than that of lactic acid (pleasant and mild dairy taste).
  • This transformation is carried out by means of the malolactic enzyme contained in the BALs of the wine (Lonvaud-Funel 1995), and can be affected by different factors that determine the growth of these bacteria such as temperature, free sulfur content, alcohol content and pH of the wine. In fact, most BALs are inhibited by ethanol and added S0 2 .
  • LABs that can be isolated in samples of musts and wines (and that belong to the genera Lactobacillus, Pediococcus, Leuconostoc, Shoeslla and Oenococcus)
  • O. oeni seems to be primarily responsible for malolactic fermentation, along with some other species such as Lactobacillus plantarum or Lactobacillus casei.
  • yeast and BAL starter cultures For the control of AF and FML there are commercial yeast and BAL starter cultures.
  • Enology products companies such as Agrovin (www.aqrovin.com, with its headquarters in Alcazar de San Juan, Ciudad Real, Spain), Lallemand (www.lallemand.com, particularly the enology division www.lallemandwine.com, with offices in California (USA), Rivas-Vaciamadrid (Madrid, Spain) and other parts of Europe), Hansen (www.chr-hansen.com), etc., manufacture and distribute starter cultures of different species and strains depending on the type of He came with whom he goes to work.
  • Oenococcus oeni cells covered with a starch gel containing the Saccharomyces cerevisiae cells that were completely deposited on the delignified cellulosic material, were immobilized on said material. After incubating the composite material at 30 e C for 24 h, it was heat dried at 35 e C for 48 h. The authors reported that heat drying was preferred to lyophilization because the cost was lower. Fermentation experiments carried out by adding this biocatalyst to grape must showed that both microorganisms acted simultaneously, obtaining the conversion of malic acid into lactic acid in 5 days and consuming sugars between 9 (in the case of glucose) and 13 days (in the case of fructose).
  • the oriental oak coal used was obtained from treated oak wood, initially, at a temperature of 300 e C for 3 hours and which, after cutting it into small fragments, was heated at a speed of 10 e C / minute at a pressure of 10-20 Pa and was subjected to a pyrolysis process at 800 e C for 1 hour in an argon gas atmosphere in an electric oven. Therefore, the process used requires a high temperature wood treatment and the use of furnaces with the possibility of modifying the atmosphere and modifying the pressure, to convert it into suitable carbon as a support, as well as the use of a yeast instead of a Bacteric acid degradation bacteria.
  • the aging stage is a long and delicate process whose objective is to confer different characters to the wine.
  • the starting point is a wine whose qualities can be improved by aging.
  • the aging process is carried out in two phases: oxidative and reducing. The first takes place in the wooden barrel, where small amounts of oxygen penetrate inside the vessel, modifying naturally and oxidatively the chemical structure of many of the components of the wine. The second is done inside the bottle and is reductive in nature. It does not penetrate practically oxygen, with the exception of very small amounts of gases that seep through the cork, so that the elements of the wine react with each other in their absence.
  • the wood gives the wine its own tannins and aromatic values, which slowly melt with the tannins of the wine. However, it is necessary to find a balance between the two partners: if the wine remained in the barrel for a long time, the rough tannins of the wood would eventually stand out over the original aromas of the wine.
  • oak which is currently the most widely used worldwide, although chestnut, cherry and acacia wood have traditionally been used.
  • the best valued woods are American oak (both the so-called American red oak, of the Quercus rubra species, as the American white oak, Quercus alba) and the so-called French oak (of the Quercus petraea or Quercus robur species ). Wood from other oak species, such as Quercus pyrenaica, is also used in Europe.
  • Quercus pyrenaica Polyrenean oak
  • Quercus petraea Quercus robur
  • Quercus robur the latter also known as carballo, cajiga or haritza
  • the quality of the wood is a determining factor in the quality of the wine obtained after aging, so it is subjected to natural, long and controlled curing processes, as well as to different degrees of roasting , so that the organoleptic characteristics of the wine are adequate.
  • the aging stage in wooden cask makes wine production much more expensive and there is currently an alternative to aging in cask that consists of the use of oak wood chips, which are put in contact with the fermentation must or with the wine already fermented. Chips, particles or wood chips are known as "chips", which are added in order to confer certain constituents coming from the mentioned wood to the wine.
  • Oak chips are permitted by legislation according to the International Wine Organization (RESOLUTION OENO 3/2005, ES-COEI-1 -MADTRO, contained in chapter I of the International Oenological Codex as the monograph entitled "Pieces of oak wood” ) and by the Community legislation of the wine sector, which already authorized its use in Council Regulation (EC) 1493/99, of 17 May, having been supplemented for application rules in Regulation (EC) No 1507/2006 e.
  • Council Regulation (EC) 1493/99 of 17 May, having been supplemented for application rules in Regulation (EC) No 1507/2006 e.
  • the addition to the wines is only authorized for pieces of wood exclusively from Quercus species, which can be used without roasting or roasting in different grades, without combustion.
  • the dimensions of the wood particles are also fixed, which must be such that at least 95% of them, expressed in weight, are retained by a sieve with 2 mm meshes.
  • Oak wood chips are porous materials, they come from a very abundant material in nature, wood, and are much cheaper than barrels, so their use in winemaking is currently widespread.
  • Oenological products companies such as Agrovin SA (www.aqrovin.com, with its headquarters in Alcazar de San Juan, Ciudad Real, Spain), Lamothe-abiet (www.lamothe-abiet.com, particularly the division of wood for oenology , http://www.lamothe-abiet-bois.com/, established in Canejan, Bordeaux, France), Arobois (www.arobois.com, Gagnac-sur- Cére, France), Christian Hansen (www.chr-hansen .com, Horsholm, Denmark) etc., have created a wide range of oak wood chips of different origins, sizes and toasts, which are commercially available. As in the case of the wood of the barrels, the chips of different roasting grade are marketed indicating the different organoleptic characteristics that they contribute, mainly
  • Kourkoutas Y et al. In its review of 2004 on various supports and immobilization techniques that have been proposed for application in the production of alcoholic beverages, mentions wood chips as one of the possible solid supports on which it has been successfully immobilize microorganisms. Specifically, it is mentioned that oak chips have been used as immobilization supports, in malolactic fermentation, for several species of Lactobacillus sp., Referring to the article by Maicas et al. from 2001 and to the article by Kosseva et al. from 1998. If these articles are reviewed, as well as the article by Janssen et al. from the year 1993 to which Maicas et al.
  • W09849264A1 discloses a method for the maturation of beer, in which the beer does not ripen (beer in which alcoholic fermentation has already occurred), after the elimination of the yeast and if subjected to a heat treatment, it is then passed to a continuous maturation bioreactor, which contains yeasts immobilized on a support.
  • Wood chips are used as support for yeasts, but among the types of chips mentioned are those of oak, but other types of wood whose use is not allowed in winemaking (beech, poplar or tropical wood) (RESOLUTION OENO 3 / 2005).
  • the preparation of the wood chips requires a previous wash and a 10% ethanol treatment, to sterilize the material, although at no time it is demonstrated that this treatment is sufficient to guarantee the absence of contaminating microorganisms.
  • the fixation of the microorganisms is carried out by a simple wet adsorption, which implies a great risk of detachment of cells from the support.
  • the request itself says that in the continuous reactor some of the yeasts are immobilized on the support, but others may be freely suspended.
  • Yeasts are the only species of microorganisms mentioned in the application.
  • yeasts used in the reactor can be conventional brewing yeasts and / or highly flocculable yeasts.
  • the possible combination of several species of microorganisms is not explicitly mentioned.
  • the application does mention that the main objective of beer manufacturing processes is the development of a product with adequate organoleptic characteristics, in addition to ensuring the fermentation efficiency itself, while the procedures for the production of specific chemical compounds, such as Ethanol, have as main objective to be efficient.
  • International patent application WO2013079797 discloses a matrix and a method for immobilizing microorganism cells in a matrix in a bioreactor that already contains the immobilization matrix when the microorganisms contact it, as well as a method to produce a fermentation product. with the bioreactor, where precisely the fermentation products that Interested in sintering are organic compounds such as ethanol or organic acids, not alcoholic beverages such as wine or beer.
  • the matrix on which microorganism cells are immobilized contains two materials, a first support material comprising a layer of porous material and a second material that retains the cells deposited on the first support material and, in addition, the layers of the first material of support and of the second material for the retention of the cells have to be arranged forming a rolled or folded structure, allowing a sufficient flow of the medium in which the cells are suspended through the immobilization matrix.
  • Wood chips are one of the possible options mentioned as one of the possible components of the matrix, specifically as a possible matrix material in which the cells are retained, while as possible support materials several plastic materials are mentioned, such as polyethylene
  • the application WO2013079797 does not contain examples demonstrating the applicability of matrices in which one of the materials are wood chips for the purposes of the invention described in said patent document, since the only experimental example described is carried out with a matrix of polypropylene and polyethylene on which cells of a single species, Propionibacterium acidipropionicii are immobilized in the reactor itself, with propionic acid being the main fermentation product of interest.
  • the only representative example of possible wood chips compatible with the invention cited in application WO2013079797 are alder chips, preferably of a size of 2x10x13 mm, which are said to give rise to high productivity and continuous flow. through column. Therefore, again, the chips whose use is suggested in this document are not of a wood whose oenological use is allowed; In addition, its use for the immobilization of microorganisms is suggested only as part of a more complex matrix, which must also include a different support material, both of which are arranged to form a folded structure.
  • the bottles that are in a horizontal position during aging are tilted with the neck down so that the yeast is deposited in the lower part of it. This helps the removal that consists of making successive turns to the bottle from time to time.
  • the duration of this second fermentation in the bottle is at least 9 months. The duration of the period of stay of the yeast inside the bottle, once the sugars are depleted, determines that a sparkling wine is qualified as a reserve, special reserve and large reserve, for the latter the regulation establishes a period that reaches five or six years , time considered maximum for the aging of this drink.
  • the sparkling fruit loses freshness and fruity aromas but gains in complexity and body as a result of prolonged contact with yeasts.
  • the preparation of the yeast for this second fermentation is very important, since the proper functioning of this second part of the cycle depends completely on it.
  • adjuvants such as bentonite are used to make it easier for yeasts to clump and settle more easily and not be suspended in the liquid.
  • the extraction of yeasts from the bottle is achieved with the procedure called degüelle.
  • the neck of the cava bottle is frozen in the area where the yeast sediment has been deposited at -4 e C, the bottle is opened and as a result of the pressure the frozen cap is fired.
  • the vacuum left in the bottle is filled with expedition liquor which is a mixture of aged wine and concentrated must (the latter is not added in the case of brut naturé).
  • Another desirable advantage is that process control is much easier than at present, because you can choose the combinations of yeasts and yeasts with bacteria more suitable for each type of wine and also, because the withdrawal of microorganisms was much more fast, which can greatly increase the diversity of the wines produced. It would also be convenient if the commercial product that could be purchased already contains the microorganisms that they wish to apply, on the same support, in a stable manner under usual conditions of commercialization, even if it is desired to apply combinations of different yeasts or, which is more complicated , of yeasts and bacteria. In addition, in order for the method developed to be of industrial interest, it would be convenient if it was carried out using products allowed by the legislation for its addition to wines and, in addition, did not require additional treatments for its use. Preferably, the cost of the products used should not be high, so that their use does not increase the cost of obtaining the wine and the price of the final product obtained.
  • the present invention provides immobilized microorganisms that are part of the microbiota associated with fermentation and that can intervene in greater or lesser degree during it, specifically lactic bacteria that synthesize the enzyme that converts malic acid into lactic acid, and / or yeasts.
  • These two types of microorganisms can be immobilized either separately or together on a support whose addition during the winemaking process is permitted by legislation and is easily accessible commercially: oak chips, preferably chips of the Quercus pyrenean species and most preferably unroasted.
  • the joint immobilization of Saccharomyces cerevisiae yeasts and lactic bacteria has the advantages that have been mentioned previously: shortening the time of the vinification process (simultaneous FM and FML), and acquisition of the characteristics of the oak, mainly.
  • the immobilized microorganisms are yeasts that carry out the alcoholic fermentation, particularly S. cerevisiae, it is also possible to use them in the second fermentation of sparkling wines, particularly the traditional sparkling cava type.
  • the advantages of this technology is that in addition to carrying out said fermentation, subsequent elimination of microorganisms (deposited in this case with the chips themselves) is facilitated and sparkling wines are obtained that have additional aromas, thus increasing the diversity of Sparkling wines in the market.
  • the chips with the immobilized microorganisms can be stored, distributed and marketed as a product that can subsequently be used by adding them at a desired stage of a process of obtaining beverages alcoholic, including discontinuous processes that are usually used for wine, especially sparkling wine.
  • the use of unroasted oak chips, compared to roasted chips results in a considerable increase in the viability of the main bacterium responsible for malolactic fermentation, O.
  • the present invention provides unroasted oak chips coated with starch, in which microorganisms are immobilized, as well as the process for obtaining them, which have the following differences and advantages over the prior art:
  • the chips of the invention are a new commercial product, since there are no commercial, unroasted, commercial oak chips with microorganisms adhered by a technique that favors their fixation (preferably lyophilization) and protected from the phenomena of detachment by the layer of starch that covers them.
  • oak chips allows their application in winemaking processes within the existing legal framework, unlike the wood chips of trees not belonging to the Quercus genus or of supports of other materials.
  • lyophilization as one of the possible options to facilitate the adhesion and fixation of microorganisms is advantageous in combination with unroasted oak chips, not only because it reduces the risk of detachment of microorganisms, but because it has an effect positive about its viability with respect to alternatives such as drying, also compatible with the present invention
  • the process of the invention allows starting from commercial oak chips, without prior treatments (except for a recommended prior sterilization process) such as delignification or carbonization
  • the process of the invention and its use with unroasted oak chips facilitates the presence of microorganisms of different species immobilized on the same chip, a possibility not considered in documents in which wood chips are also used as support and, in particular, allows the immobilization on the same chip of yeasts and bacteria, such as bacteria with enzymes to catalyze the malolactic fermentation, maintaining these viability sufficient for said fermentation to take place, and facilitating the simultaneous incorporation into a process of vinification of yeasts responsible for fermentation alcoholic and / or other yeasts and bacteria capable of producing malolactic fermentation
  • the chips of the invention in which the microorganisms are immobilized on an oak chip as a support, facilitate the elimination of the microorganisms when desired or considered to have fulfilled their function, unlike some other supports such as coal dust
  • Oak chips confer pleasant organoleptic properties, both color and taste and aroma, to beverages into which production is incorporated, which is maintained in the chips of the invention.
  • the present invention relates to said oak chips, to process for obtaining it and its use in the wine preparation process, either during winemaking as such or during the second fermentation of sparkling wines.
  • a first aspect of the present invention is a method for obtaining oak chips or chips comprising vinification microorganisms immobilized on their outer surface and / or that of their pores comprising the steps of: a) incubating the oak chips with cells of at least one of the species of microorganisms to be immobilized on them; b) mixing the chips with already adhered microorganisms obtained in step a) with a starch gel, which optionally has been previously mixed with cells of at least one of the microorganism species to be immobilized on the chips; c) immobilize the microorganisms on the chips by drying or lyophilization; characterized in that unroasted oak chips are used.
  • the invention in a second aspect, relates to an unroasted oak chip characterized in that it comprises immobilized vinification microorganisms on its outer surface and / or on the surface of its pores.
  • the chips obtained by the method of the present invention are included.
  • the invention relates to the use of the oak chips of the invention in the production of an alcoholic beverage, especially a wine, either during the vinification of a must or in the process of the second fermentation of a Sparkling wine, in this case preferably cava type, although they can also be used in the production of alcoholic beverages other than wine, such as cider.
  • an alcoholic beverage especially a wine
  • a Sparkling wine in this case preferably cava type, although they can also be used in the production of alcoholic beverages other than wine, such as cider.
  • Fig. 1 shows photographs of oak chips
  • Panel A life-size photographs of different oak chips: a) natural Iberian (unroasted Quercus pyrenaica chips), b) high roasted Iberian (Quercus pyrenaica chips subjected to high roasting), c) medium roasted Iberian (chips Quercus pyrenaica subjected to medium roasting), d) French medium roasting (mixing chips of Quercus petreae and Quercus robur with medium roasting)
  • Panel B Electron microscopy photographs of unroasted Iberian oak chips, where its porosity is observed.
  • Fig. 2 corresponds to electron microscopy photographs showing S. cerevisiae and O. oeni cells co-immobilized in unroasted Iberian oak chips fixed: panel a) by drying, panel b) by lyophilization.
  • Fig. 3 shows graphs of the consumption of sugars (glucose and fructose) and malic acid during the vinification process from curves that show the variation of its concentration as a function of time:
  • Fig. 3A results obtained by sequential inoculation of free S. cerevisiae and O. oeni cells (traditional method). It also includes the kinetics of appearance of lactic acid, glycerol and ethanol from its concentration in grams per liter or, in the case of ethanol, the percentage detected over the days.
  • - Fig. 3B results obtained by simultaneous inoculation of S. cerevisiae and O. oeni, either in the form of free cells or co-immobilized in unroasted Iberian oak chips, as indicated in brackets next to the legend of each curve.
  • Fig. 4 shows the consumption of sugars and malic acid during the winemaking process by inoculation of co-immobilized S. cerevisiae and L. plantarum in unroasted Iberian oak chips.
  • Fig. 5 shows the consumption of sugars and the formation of ethanol during the second fermentation of bottled cava, A) by inoculation of S. cerevisiae free cells and B) immobilization of S. cerevisiae immobilization in Iberian oak chips without toast.
  • Fig. 6 corresponds to photographs of the stages of elaboration of a sparkling wine type cava in which the second fermentation takes place by inoculation of S. cerevisiae immobilized in unroasted Iberian oak chips, in which it is observed, A) the Rhyming position of the bottles during aging, B) bottles with the frozen neck for its disgorgement and C) sparkling wine type champagne finish of transparent appearance after the disgorgement.
  • the present invention results from a project whose objective is the development of co-immobilized starter cultures of yeasts and malolactic lactic acid bacteria for the simultaneous realization of AF and FML in wine and the immobilization of yeasts for the second fermentation of the cava in a support Suitable for use in winemaking processes, such as oak wood chips, the use of which is permitted by law.
  • the invention relates to starch-coated oak chips comprising vinification microorganisms immobilized on their outer surface and / or on the surface of their pores and on the outer starch film, to the use of said chips in the production process of alcoholic beverages, particularly in the process of producing a wine, and the method for obtaining said chips comprising the steps of: a) incubation of cells of at least one of the microorganism species to be immobilized with the oak chips; b) coating the chips in which there are already microorganisms adhered with the starch gel, contacting and mixing the chips with the starch gel; said starch gel, optionally, may have previously been mixed with cells of at least one the species of microorganisms to be immobilized, which species may be the same as in step a) or a different one; c) immobilization of the microorganisms on the chips by drying or lyophilization.
  • chips, particles or wood chips are called "chips", which are added with the purpose of conferring certain constituents from the mentioned wood to the wine.
  • chip is preferred, in addition to being widely extended among those skilled in the art, for the linkage that said term has with its use in vinification and with the definition of wood chips or chips whose use is allowed by legislation, pieces that, as discussed above, can only be added to wines if they come exclusively from Quercus species.
  • chips can be natural (unroasted), which is especially preferred in the present invention, or roasted in different degrees, without combustion, and whose dimensions must be such that at least 95% of the pieces or chips, expressed in weight, are retained by a sieve with 2 mm meshes.
  • wood chips or chips called chips meet said legislative requirements.
  • said chips if treated according to the method of the present invention, can effectively serve as a support for microorganisms involved in the wine production process, either in the vinification of table wines or of sparkling wines, and, in the case of the latter, especially in their second fermentation.
  • starch-coated oak chips are a suitable support for immobilizing different microorganisms, both yeast and bacteria, without the need for prior processing of the chips such as delignification, process which is carried out in previous documents referring to the use of cellulosic materials as a support for microorganisms, whether the microorganisms are only lactic bacteria such as O.
  • immobilization matrices that are simpler in structure can be considered than those described in WO2013079797A1, which involve the presence of two materials (one porous support and one for cell retention) and which, in addition, must be arranged forming a rolled or folded structure;
  • the preparation of the chips of the present invention in addition to not being carried out by simple wet adsorption, is prior to their introduction into the industrial process in which they are to be used and independent of the reactors used in said processes, allowing a prior preparation, an independent commercialization and, after its acquisition, the possible application to different processes, as desired.
  • they are compatible with the usual processes of wine production, particularly those of sparkling wines, which does not happen with the similar supports described in the documents W09849264A1 or WO2013079797A1, designed for continuous processes and whose obtaining is integrated into said processes.
  • the chips on which microorganisms are immobilized are oak chips, that is, of a species of the genus Quercus.
  • the oak chips give the wine organoleptic properties of the wood. Its use as an alternative to barrel aging reduces the process time and avoids investment in barrels and therefore reduces the costs of the winemaking process (Gómez Garc ⁇ a-Carpintero et al. 2012).
  • oak chips are natural and accepted supports compared to other supports studied and with difficult acceptance in wine such as alginate, silica, polyacrylamide , apple, etc. (Rossi and Clementi 1984; Fumi et al. 1987; Kourkoutas et al. 2006; Callone et al. 2008).
  • Oak chips as mentioned above, are an accepted material for use in wine according to the International Wine Organization (OENO RESOLUTION 3/2005).
  • unroasted chips especially Quercus pyrenaicá
  • the unroasted Iberian oak chips also enhance the floral and varietal aromas of the wine, have high antioxidant capacity and enhance the color stabilization due to its high concentration of a certain tannin, elagitannin, which represents an important tannic reinforcement in the mouth increasing unctuousness and volume.
  • the improvement in cell viability achieved by the use of unroasted oak chips is especially important when you want to immobilize malolactic bacteria, microorganisms whose viability is particularly affected when coming into contact with oak chips with varying degrees of roasting.
  • the use of unroasted oak chips facilitates that malolactic bacteria can be immobilized on them and that these microorganisms maintain its viability and can produce malolactic fermentation when added during a winemaking process as part of chips of the present invention, avoiding having to resort to microorganisms that are not the usual ones to carry out said fermentation, such as yeasts used by Hong et al . in the aforementioned document, which may lead to the appearance of compounds whose influence on the organoleptic characteristics is unknown and which may eventually be negative.
  • the chips of the invention can simultaneously contain yeasts and bacteria, in particular malolactic bacteria, a rare combination in the state of the art, but which makes it possible for the chips of the invention to be used in winemaking processes in such a way as to produce jointly alcoholic fermentation and malolactic fermentation, a possibility that is considered advantageous during wine production, as discussed above.
  • the starch-coated oak chips of the present invention comprise vinification microorganisms immobilized on their outer surface and / or on the surface of their pores, as well as on the starch cover itself.
  • any microorganism that is involved in this process is called the vinification microorganism, including not only yeasts that are capable of giving rise to alcoholic fermentation or the bacteria that transform malic acid into lactic acid ( BAL) but to other microorganisms such as yeasts or bacteria that, regardless of whether or not they are capable of giving rise to alcoholic or malolactic fermentation, can produce substances that modify or improve the organoleptic profile in the conditions of production of a wine, including those of aging or sparkling.
  • the chips of the present invention can be used not only to facilitate alcoholic and malolactic fermentation, while imparting to the wine aromas of the wood, but also to confer wine aromas produced by non-Saccharomyces yeasts.
  • the invention contemplates that the vinification microorganisms are vinification yeasts.
  • S. cerevisiae is used as the starter culture to carry out AF, whereby a preferred embodiment of the invention implies that microorganisms that at least comprise yeasts of said species are immobilized on oak chips.
  • examples of the present application include tests in which oak chips are used in which only S. cerevisiae yeasts are immobilized, also describing their preparation procedure, a process in which the yeast cells are first incubated with the chips, then I know Mix the chips in which the yeast cells are attached with starch gel (in this case, wheat), then immobilize the microorganisms.
  • This procedure is also preferred when it is desired to immobilize other vinification yeasts, which may be of a single species or of several.
  • other non-Saccharomyces yeast species such as Hanseniaspora uvarum (also known as KIoeckera apiculata), which play an important role in winemaking, since they provide greater aromatic complexity to the wine and therefore they may be interesting as starter cultures, as also shown in trials included in the Examples of the present application.
  • the microorganisms immobilized in the chips and those immobilized in the method of they are yeasts capable of carrying out alcoholic fermentation, of the genus Saccharomyces and / or non-Saccharomyces, but also other yeasts that synthesize other substances that can confer on the wine desired organoleptic characteristics, with preference for those species that are known to they have been isolated from musts, wines in preparation or from the containers in which the winemaking process is carried out.
  • Co-immobilization of vinification yeasts and malolactic lactic acid bacteria is also within the scope of the invention.
  • lactic bacteria that are capable of carrying out malolactic fermentation that is to say the transformation of malic acid into lactic acid, have been named, so they have to be able to synthesize enzymes capable of catalyzing said reaction under the conditions in which the vinification process takes place, at least during a period of it.
  • Oenococcus oeni which is the bacterium generally used as a starter culture to carry out the FML. It is a possible BAL for the purposes of the invention and, in fact, co-immobilization S.
  • microorganisms that co-immobilize on the starch-coated oak chips of the present invention comprise vinification yeasts and malolactic lactic acid bacteria
  • they are bacterial cells those that adhere initially on the chips, mixing and incubating with agitation a sample of biomass of bacterial cells with the chips and with a liquid medium that includes either a culture medium for the bacteria or, in the case that it is going to proceed to lyophilization, a cryoprotectant compound; ii) the yeasts are mixed with the starch gel prior to mixing with the chips; iii) the chips in which the bacterial cells have been immobilized are gradually added to the mixture of yeast starch gel until the gel is not able to embed more immobilized; iv) finally the final immobilization of both microorganisms occurs after mixing the starch gel previously mixed with yeasts and the chips with previously immobilized bacterial cells.
  • the chips when the chips are mixed with the starch gel, the latter has already been previously mixed with yeasts, while the chips already have bacteria adhered thereon when said mixture is produced.
  • the process in its most general definition and, specifically, in regard to the first stage of the process, the incubation, in which microorganism cells are mixed and kept in contact with the oak chips for a while , it is important that the cells are homogenized well with the chips so that they are adhered to them, but it is not necessary to prolong the incubation time so that there is cell replication because, as a culture with a high concentration of cells is used (concentrated culture referred to in several points of the application as "biomass”), the cells have no room to grow.
  • a suitable incubation time is considered a value between 30 minutes to 1 hour, with special preference for incubation for 30 minutes, as in the tests of the Examples of the present application, since prolonging it up to 1 hour did not give rise to observing differences in the results.
  • the incubation temperature it must be chosen taking into account the microorganisms that you wish to immobilize on the chips. Since the present invention is preferably directed to immobilized microorganisms that intervene or are present during the vinification process, or in a possible second fermentation, the range of 10-15 e C to 30 e C may be a suitable reference range for the choice of said temperature.
  • yeast in particular Saccharomyces cerevisiae
  • 30 e C so as not to compromise its viability.
  • 28 e C is used, which is a usual temperature for the microorganisms used in said Examples, compatible with all of them.
  • any starch with gelatinizing properties can be used, provided that the temperature range within which its gelatinization occurs, which varies from one to the other, is taken into account.
  • Wheat starch is preferred, whose total gelatinization occurs in the middle range of 80-90 e C, even 80-95 e C, although other natural starches such as ordinary corn starch (containing approximately 27 can also be used) % of amylose and have an average gelatinization range of 70-72 e C) or potato (average gelatinization interval: 60-75 e C).
  • Starches called "waxy”, which come from some natural varieties of barley, corn, rice and sorghum and which, due to their low amylose content, are less able to form gels are less recommended.
  • modified starches capable of gelatinization can also be used, also taking into account that, for the purposes of the invention, starches whose use is admitted in food products are preferred. For information in this regard, please consult the Royal Decree 142/2002 of 1 February and Regulation (EC) No 1333/2008 and the European Parliament and of the Council of 16 December 2008 on food additives.
  • the gelatinization of the starch must first be carried out, whereby a step of heating it in water to the appropriate temperature for that said gelatinization occurs: the collection of water by the starch granules, which increases as the temperature increases, resulting in the granules swelling, at the exit of amylose chains from the starch granules, the disintegration of the granules and the formation of a paste.
  • temperatures in the range of 80 e C-95 e C, for example 90 e C may be suitable, as in the Examples of the present application.
  • a gel an elastic solid that involves a two-phase system with a solid continuous phase of amylose polymers that forms a three-dimensional network that retains a liquid dispersed phase.
  • it may be adequate cooling, for example, up to 45 and C, as in the case of Examples of the present invention.
  • the starch gel is then mixed either with the microorganism cells directly (for example, with yeast cells, as in the cases of co-immobilized bacteria and yeast described later in the Examples of the present invention), prior to mixed with the chips, or with chips on which microorganisms have already been adhered (microorganisms that are preferred to be BALs in cases of immobilization of lactic bacteria and yeasts, but which can also be yeasts, as in Example 4 of this application).
  • This stage may be shorter, almost instantaneously, than the initial stage of incubation of microorganism and chip cells, since it seeks to homogenize the starch (mixed or not previously with microorganisms) with the chips that already have attached microorganisms.
  • the present invention considers drying and lyophilization.
  • the method of the present invention preferably comprises a first drying of the bacteria after incubation with the chips, prior to mixing with the yeasts, and a second final drying for the joint fixation of both microorganisms after mixing the wheat starch gel with yeasts and the chips with immobilized bacterial cells.
  • the drying can take carried out, for example, as indicated in the initial methodological subsection included in the Examples section of the present invention, both the first drying of the bacteria and the second drying for the joint immobilization of bacteria and yeasts are performed at 35 e C for 24 hours.
  • lyophilization is preferred. As shown in tests of the Examples of the present application, lyophilization, in combination with the method of the present invention, allows to achieve greater cell viability than other techniques such as drying, making it the preferred technique for carrying out The method of the present invention.
  • the fixation of both microorganisms occurs in a single lyophilization stage, which takes place once the chips have been mixed with the starch gel and, thus, have already been mixed with all the microorganisms that must remain immobilized on the same, which reduces processing time and reduces cell death compared to studies in which two consecutive drying stages are applied (see, for example, Servetas et al. 2013, where preference is shown for drying over to lyophilization).
  • the production costs of the co-immobilized are reduced by simultaneously freeze-drying 2 starter cultures in a single support, avoiding 2 separate drying stages.
  • lyophilization is a procedure to favor adhesion that is preferable over heating and that provides advantages over it, especially given that documents that use other cellulosic materials such as support, as is the case of the article by Servetas et al. from 2013, there was a preference for drying, also in two stages, with respect to lyophilization.
  • the starch-coated oak chips of the present invention will also preferably comprise a cryoprotectant.
  • cryoprotectant it is convenient to take into account the type of microorganism or microorganisms that want to lyophilize.
  • BAL monosodium glutamate
  • yeasts in general glucose is common.
  • monosodium glutamate is preferred as a cryoprotectant to carry out the lyophilization fixation process in the case of co-immobilized yeast and BAL production, because the latter are more sensitive to wine conditions than yeasts and It is better to protect them more.
  • this cryoprotectant increases viability and cell activity during lyophilization (Zhao and Zhang 2005; Zhao and Zhang 2009).
  • Other cryoprotectants can also be used, for example, as glucose has already been indicated.
  • yeasts e.g. Saccharomyces cerevisiae
  • yeasts e.g. Saccharomyces cerevisiae
  • the viability of yeasts is similar both in the tests performed with the co-immobilized with BAL, where the cryoprotectant was monosodium glutamate, as in the tests performed with the chips of Example 4, chips in which the yeasts were the only microorganisms fixed and in which glucose was used as a cryoprotectant.
  • monosodium glutamate can be used both in one and another case, a different cryoprotectant not being necessary depending on the microorganism.
  • the immobilized and co-immobilized starter cultures on starch-coated oak chips of the present invention have a clear application in the wine production process, either by adding them during the winemaking process of a must (where they can be add both to the unfermented must, as when the fermentation process has begun, at some point along the same), as in other processes to which the wine can be subjected as is the second fermentation that takes place in wines sparkling, as demonstrated in Example 4 of the present application.
  • chips in which only yeasts have been immobilized preferably Saccharomyces cerevisiae, such as those used in said Example 4; such chips may also be a possible option for the fermentation of white wines, in many of which FML does not usually take place or is less important than in red wines in general.
  • the immobilization of yeast on oak chips also has the advantage that it is a more accepted material than monosodium alginate in oenology because it is commonly used, although for purposes other than those described here (Gómez Garc ⁇ a-Carpintero et al. 2012).
  • Example 4 describes the application in a second fermentation process in the bottle such as those that take place in sparkling cava type wines obtained by the traditional method, adding the chips to the bottle at the stage in which it is traditionally added to the base wine the draft liquor, is perfectly compatible with the present invention the use for the second alcoholic fermentation of other sparkling wines, such as those produced in large containers or tanks, in which the wine is Bottles when it is already gasified, production method sometimes referred to as charmat or granvas.
  • cider can be of special interest, since apples are fruits rich in malic acid and the elaboration of a quality cider requires that malolactic fermentation takes place, which, in a traditional way, takes place once the alcoholic fermentation is finished; the addition of coinmobilized yeast and BAL starter cultures on starch-coated oak chips, preferably unroasted, could also help shorten the cider production process.
  • the use of the oak chips of the invention that is, the starter cultures of microorganisms immobilized on oak chips, in the production process of an alcoholic beverage in general, is also included within the scope of the present invention. with particular preference for wine and cider.
  • wine In the specific case of wine, all the particularities that have been discussed in the previous paragraphs are included, including with the use for the second fermentation of sparkling wines.
  • the open design of the system allows the planning of different types of co-immobilizations (bacteria-bacteria), (bacteria-yeast), (yeast-yeast) or simple immobilizations, which can be designed according to the preferred application and specific effects that you wish to obtain.
  • yeast strain Saccharomyces cerevisiae Enolab 5021
  • a yeast suitable for carrying out AF in must or the second fermentation of cava H. uvarum CECT 1444 (yeast used for the aromatic improvement of wines) and bacterial strains Oenococcus oeni Enolab 5003 (Berbegal et al. 2015)
  • Lactobacillus plantarum Enolab 4608 (Lucio 2014), which are BAL selected to perform the FML in must or wine, isolated all during the fermentation of the must or wine.
  • oak chips from the Iberian Peninsula ⁇ Quercus pyrenaica were used without roasting (Spirit NATURE, Agrovin SA), with medium roasting (Spirit STRUCTURE, Agrovin SA) and with high roasting (Spirit ELEGANCE, Agrovin SA), in addition of French oak chips ⁇ Quercus petraea and Quercus robur) with medium roasting (Spirit CLASSIC, Agrovin SA).
  • the chips were sterilized by dry heat (160 e C, 2 h) prior to use.
  • Fig. 1 shows a photograph of said chips.
  • the immobilization of O. oeni on oak chips was performed.
  • the bacterial biomass was generated in OPM culture medium ⁇ Oenococcus Production Medium) (Berbegal et al. 2015) until a concentration of 2x10 9 CFU / mL was reached.
  • the culture was centrifuged at 8000 rpm for 15 min (Heraeus Multifuge 1 SR) and the supernatant was removed.
  • a stirring incubation (120 rpm / min) of a mixture composed of 7% cellular biomass, 15% oak chips and 78% OPM for 30 min at 28 e C was carried out to promote the adherence of the bacteria to the support .
  • yeast biomass was generated in GPY culture medium ⁇ Glucose Yeast Extract Peptone, Khondar et al., 2002: glucose 40 g / l, peptone 40 g / l, yeast extract 3 g / l) liquid until reaching a concentration of 5x10 8 CFU / mL.
  • the culture was centrifuged at 8000 rpm for 15 min (Heraeus Multifuge 1 SR) and the supernatant was removed.
  • a mixture of wheat starch (Fluka, currently available from Sigma Aldrich with code S5127) was made in 8% water and heated to 90 ° C until a gel consistency was achieved. When the temperature dropped to 45 and C, the biomass mixture was added to the yeast 13% To this mixture was added gradually the immobilized O. oeni until the gel was not able to imbibe more immobilized. Subsequently, a second drying of the immobilized with both microorganisms was carried out at 35 e C for 24 h. The dried starter culture was stored at 4 e C in the dark under cover of air. The immobilization of the microorganisms was checked by electron microscopy (JSM-6300 scanning electron microscope of JEOL).
  • a stirring incubation of a mixture composed of 7% cellular biomass, 15% oak chips and 78% cryoprotectant (0.067 mM L-sodium glutamate, Panreac) was carried out for 30 min at 28 e C to promote adhesion of Bacteria to the support.
  • the immobilization of yeasts (S. cerevisiae or K. apiculatá) was performed on the bacterial fixed assets.
  • the cell biomass was previously generated in liquid GPY culture medium until it reached a concentration of 5x10 8 CFU / mL.
  • the culture was centrifuged at 8000 rpm for 15 min (Heraeus Multifuge 1 SR) and the supernatant was removed.
  • a mixture of wheat starch (Sigma Aldrich S5127) is performed in water 8% and heated at 90 ° C until a gel consistency, and when the temperature dropped to 45 and C was added biomass mixture 13% yeasts. To this mixture was added gradually immobilized with O. oeni until the gel was not able to embed more immobilized. Subsequently, water removal was carried out by sublimation. For this purpose, the fixed assets were frozen, containing both microorganisms at -20 ° C for 6 h. The lyophilization was carried out for 27 h under vacuum conditions (15.9 millimeters) in a Virtis Sentry equipment. The lyophilized starter culture was stored at 4 e C in the dark under cover of air. The effectiveness of the immobilization was checked by electron microscopy (JSM-6300 scanning electron microscope of JEOL).
  • Yeast S. cerevisiae Enolab 5021 was grown in liquid GPY culture medium to reach a concentration of 2x10 9 CFU / mL. The culture was centrifuged at 8000 rpm for 15 min (Heraeus Multifuge 1 SR) and the supernatant was removed. The Stirring incubation of a mixture composed of 7% cellular biomass, 15% oak chips and 78% cryoprotectant (15% glucose, Panreac) for 30 min at 28 e C to promote yeast adhesion to the support.
  • the percentage of viable cells and dead cells was calculated using the LIVE / DEAD BacLight bacterial Viability Kit (Invitrogen). To do this, 0.1 g of immobilized in 100 ⁇ of distilled water were resuspended and 0.3 ⁇ of the mix (1 v solution A: 1 v solution B) of the kit was added. It was incubated for 20 min in the dark and the samples were observed at 100X with immersion oil in a fluorescence microscope (Leica). Viable cells have green fluorescence and dead cells have red fluorescence. Taking into account the percentage of viable cells, the number of viable cells per gram of fixed assets was calculated.
  • a must of the Tempranillo variety (glucose 65 g / L, fructose 65 g / L, L-malic acid 3.0 g / L, pH 3.5) was used. Three inoculation strategies in must were followed. The vinifications were made in triplicate at 25 e C.
  • S. cerevisiae was inoculated in a final concentration of 1 x 10 6 CFU / mL. After completion of AF, O. oeni was inoculated in a final concentration of 1 x 10 6 CFU / mL.
  • Simultaneous AF and FML using free yeast cells and bacteria S. cerevisiae and O. oen / ' were co-inoculated in a final concentration of 1 x 10 6 CFU / mL.
  • the base wine (Macabeo and Chardonnay, 1 1% ethanol (v / v)) was distributed in transparent cava bottles, to which was added a circulation liquor (12 g / L of glucose and 12 g / L of fructose).
  • the bottles were inoculated with the yeast immobilized in unroasted Iberian oak chips (1 g / L corresponding to approximately 2x10 6 CFU / mL) or with a free cell culture (2x10 6 CFU / mL) and aging of 9 months in bottle.
  • the experiments were performed in triplicate.
  • Sugar consumption and ethanol formation during fermentation was analyzed using an HPLC (Agilent 1200 series), AMINEX HPX-87H (BIORAD).
  • the mobile phase consisted of a solution of 0.75 ml of H 3 P0 4 at 85%.
  • the formation of aromatic compounds was carried out by means of a gas chromatography device. The tasting was carried out through a panel of 10 expert tasters.
  • Example 1 Viability and effectiveness of the immobilization of S. cerevisiae v O. oeni in the different types of oak chips.
  • oak chips from the Iberian Peninsula were used without roasting, with medium roasting and high roasting.
  • French oak chips ⁇ Quercus petraea and Quercus robur) with medium toast.
  • the American oak Quercus alba was discarded due to its low porosity.
  • the French and Iberian oak are very porous, favoring greater cell absorption.
  • the immobilization and arrangement of the yeast cells (S. cerevisiae or K. apiculatá) and the bacterial cells (O. oeni or L. plantarum) co-immobilized were checked by electron microscopy. Two techniques of adhesion of the microorganisms to the chip for obtaining the co-immobilized yeast-bacteria, drying and lyophilization were studied. As an example, in Fig. 2 it can be seen that S. cerevisiae and O. oeni cells are adhered inside the oak chip and covered by a layer of starch whether they are fixed by drying or by lyophilization. The yeasts remain in the coating film itself, while the bacteria remain below. The results shown correspond to an initial incubation time of the chips with microorganism cells of 30 minutes, but incubation for 1 hour did not lead to differences.
  • Example 3 Evaluation of the immobilization of other microorganisms of oenological interest
  • H. uvarum Immobilizing a yeast non-Saccharomyces present in early stages of the vinification, H. uvarum showed that the concentration of viable cells after lyophilization was 2.5x10 8 - 3x10 8 CFU / g.
  • the immobilized yeast was deposited without the help of adjuvants such as bentonite as can be seen in Fig. 6A.
  • adjuvants such as bentonite
  • Fig. 6B the frozen plug formed by the yeasts and the immobilization support is observed.
  • the cava ready to be consumed, presented a transparent appearance and no remains of the immobilized starter culture (Fig. 6C).

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Abstract

L'invention concerne des rabotures avec des microorganismes, leur préparation et leur utilisation. Le procédé de la présente invention permet d'obtenir des cultures de microorganismes immobilisés en rabotures ou en copeaux de chêne recouverts d'amidon. Il utilise des copeaux de chêne sans délignification, à usage autorisé dans les vins. Les microorganismes restent immobilisés dans les pores des rabotures ou des copeaux, de préférence les espèces de chêne de haute porosité et, plus particulièrement, les copeaux non grillés, qui donnent lieu à une meilleure viabilité des microorganismes, avec l'immobilisation par lyophilisation. Les copeaux obtenus sont particulièrement utiles dans l'élaboration du vin, parce qu'ils donnent simultanément les caractéristiques du bois et interviennent dans les fermentations. Les co-immobilisés de levures et bactéries malolactiques permettent la fermentation alcoolique et malolactique simultanées, tout en accélérant la production. Les levures de fermentation alcoolique immobilisées dans ces copeaux peuvent s'appliquer à la seconde fermentation de mousseux, facilitant l'élimination des levures et apportant de nouvelles caractéristiques organoleptiques.
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ES2748189A1 (es) * 2018-09-13 2020-03-13 Montagut Lalauze Jose Alain De Metodo para la aromatizacion de bebidas alcoholicas
ES2932808A1 (es) * 2021-07-20 2023-01-26 Univ Cadiz Dispositivo de flotacion para la regeneracion artificial del velo de flor

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108251411A (zh) * 2018-02-13 2018-07-06 南阳师范学院 一种利用橡木片制备葡萄酒用固定化酵母的方法
ES2748189A1 (es) * 2018-09-13 2020-03-13 Montagut Lalauze Jose Alain De Metodo para la aromatizacion de bebidas alcoholicas
ES2932808A1 (es) * 2021-07-20 2023-01-26 Univ Cadiz Dispositivo de flotacion para la regeneracion artificial del velo de flor

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