AU641360B2 - Process for preparing micro-organisms incorporated within substantially dehydrated gels, gels obtained and their use in the preparation of fermented beverages - Google Patents

Process for preparing micro-organisms incorporated within substantially dehydrated gels, gels obtained and their use in the preparation of fermented beverages Download PDF

Info

Publication number
AU641360B2
AU641360B2 AU39612/89A AU3961289A AU641360B2 AU 641360 B2 AU641360 B2 AU 641360B2 AU 39612/89 A AU39612/89 A AU 39612/89A AU 3961289 A AU3961289 A AU 3961289A AU 641360 B2 AU641360 B2 AU 641360B2
Authority
AU
Australia
Prior art keywords
micro
organisms
gel
concentration
drying
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.)
Ceased
Application number
AU39612/89A
Other versions
AU3961289A (en
Inventor
Jacques Beaujeu
Charles Divies
Frederic Herault
Pascal Lenzi
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.)
Champagne Moet and Chandon SA
Original Assignee
Champagne Moet and Chandon 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
Priority to FR8809249A priority Critical patent/FR2633937B1/en
Priority to FR8809249 priority
Application filed by Champagne Moet and Chandon SA filed Critical Champagne Moet and Chandon SA
Publication of AU3961289A publication Critical patent/AU3961289A/en
Application granted granted Critical
Publication of AU641360B2 publication Critical patent/AU641360B2/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00Microorganisms, 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
    • C12N1/04Preserving or maintaining viable microorganisms
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12CBEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
    • C12C11/00Fermentation processes for beer
    • C12C11/09Fermentation with immobilised yeast
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12GWINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
    • C12G1/00Preparation of wine or sparkling wine
    • C12G1/02Preparation of must from grapes; Must treatment and fermentation
    • C12G1/0203Preparation of must from grapes; Must treatment and fermentation by microbiological or enzymatic treatment
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/04Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02EREDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00Technologies for the production of fuel of non-fossil origin
    • Y02E50/10Biofuels, e.g. bio-diesel

Description

OPI DATE 05/02/90 AOJP DATE 22/03/90 APPI N- 1D 39612 89 PCT NUMBER PCT/FR89/00355 DEMANDE INTERNATIONALE PUBLIEE. EN VERTU DU TRAITE DE COOPERATION EN MATIERE DE BREVETS (PCi) Classification Internationale des brevets 5 (11) Numiro de publication Internationale: WO 90/00602 CI2N1/04Al (43) Date de publication Internationale: 25 janvier 1990 (25.01.90) (21) Numilo de Ia demnande Internationale: PCT/FR89/00355 (74) Mandataires: PORTAL, Gerard etc. Cabinet Beau de Lo- (22) Date de dip6t international: 6juillet 1989 (06.07.89) ni,5,redAsramF-08Prs(11) (81) Etats disignis: AU, BR, DK, JP, KR, US.
Donnies relatives a In priorit6: 88/09249 7 juillet 1988 (07.07.88) FR Publiie A;'ec rapport de recherche intemnationac.
(71) Diposant (pour tous les Etats d~sign&s sauf US): CHAMPA- Avant I'expiration du dMai pr~vupour la mod ification des GNE MOET CHANDON [FR/FR]; 20, avenue de revendications, sera republie si de telles mnodifications sorn Champagne, F-5 1205 Epernay C~dex (FR1). repies, (72) Inventeurs; et Inventeurs/Diposant (US seulement) :DIVIES, Charles [FR/FRk]; 90, rue des P~joces, F-21000 Dijon LEN- 2 1000 Dijon BEAUJEU, Jacques [FR/FR]; 11, rue6 4 Lenez, Magenta-Noum~a, Nouvelle Cal~donie (FR1).
HERAULT, Frid&ric [FR/FR]; 16, rue de ]a Blanchetikre, F-44240 La Chapel le-su r- Erdre (FR).
(54)Title: PROCESS FOR PREPARING MICRO-ORGANISMS INCORPORATED WITHIN SUBSTANTIALLY DEHY- DRATED GELS, GELS OBTAINED AND THEIR USE IN THE PREPARATION OF FERMENTED BEVE-
RAGES
(54)Titr e: PROCEDE DE PREPARATION DE MICRO-ORGANISMES INCLUS DANS DES GELS SENSIBLEMENT DESHYDRATES, GELS OBTENUS ET LEUR UTILISATION POUR LA PREPARATION DE BOISSONS
FERMENTEES
(57) Abstract' In a process for preparing micro-organisms incorporated within at least partially dehydrated gels, a) the micro-organisms are incorporated in a polymer solution capable of forming a gel; b) the solution containing the micro-organisms is gelled to form a gel in which the micro-organisms are incorporated; c) the gel in which the micro-organisms are incorporated is dried to obtain at least partially dehydrated gel. The process is characterized in that gels in which micro-organisms having a high concentration of hydrophilic substances are prepared and then subjected to said drying step. This is preferably effected by soaking the gel in a solution of hydrophilic substances having said high concentration until equilibrium is attained, and then subjecting the gel to said drying step, The gels obtained, in which the micro-organisms are incorporated, have a high concentration of hydrophilic substances and enhanced viability after rehydration.
(57) Abrigi L'invention concerne un proc~d& de preparation de micro-organismes inclus dans des gels au momns partiellement d~shydrat~s. Ce proc6d& comprend: a) l'inclusion de micro-organismes danis une solution de polymire susceptible de se transformer en gel; b) Ia g~lif ication de cette solution contenant les micro-organismhes de mani~re Ai former un gel incluant les micro-organismes; et c) le s~chage de ce gel incluant les micro-organismes, pour obtenir un gel au momns partiellement dishydrati, caract~ris6 en ce qu'on prepare des gels incluant les micro-organismes ayant une forte concentration en substances- hydrophiles que l'on soumnet ensuite ladite 6tape de s~chage. De pr~ffrence, ceci est obtenu par trempage du gel dans une solution de substances hydrophiles ayant ladite forte concentration, jusqu'zi l'~quilibre, puis en soumnettant le gel at l'6ape de s~chage pr~cit~e. Les gels obtenus, incluant des micro- organismnes, ayant une forte concentration en substances hydrophiles prisentent une viabilit amilior~e apris r hydration. Process of preparation of micro-organisms enclosed in appreciably dehydrated gels, gels obtained and their use for the preparation of fermented drinks.
The present invention relates essentially to a process for the preparation of micro-organisms enclosed in appreciably dehydrated gels, gels obtained and their use for the preparation of fermented drinks.
The present invention was made within the framework of the laboratory of microbiology of the Ecole Nationale Superieure de Biologie AppliquBe a la Nutrition et a 1'Alimentation (ENSBANA).
In the state of the prior art, it is known that immobilized micro-organisms can be used in the production of fermented drinks such as wine and beer (FR-A-2 320 349 and FR-A-2 359 202). Their use has also been suggested for classical champagnization (FR-A-2 432 045) as well as for the manufacture of sparkling drinks with a variable degree of alcohol (FR-A-2 601 687). These publications, as well as others (in particular JP-A-57-150 385 or EP-A-173 925) have emphasized the performances of reactors with immobilized cells.
These techniques have made possible the carrying out of fermentations with mixtures of micro-organisms of different categories (mixture of lactic acid bacteria and mixture of yeast).
However, the implementation of the process on an industrial scale came up against the difficulty of having available particles capable of being preserved for a long period.
Micro-organisms in the enclosed form may be used without an appreciable drop of activity over long periods when the nutrition of the micro-organisms concerned is respected.
It has been possible to note a toxicity of the products of fermentation which leads to a partial ageing of the cells (article by DIVIES et al. in Annales de microbiologie, 1977, pages 349-358).
It was thus preferred in this particular case to use a battery of reactors of defined life time and to carry out the partial renewal of the microbial particles in a programmed manner.
Problems of mechanical resistance of the gel have also managed to arise and are described in JP-A-57-150 385.
It thus appears crucial at the industrial level to plan the elaboration of the particles for inclusion of the micro-organisms and to centralize the production of them. It is also necessary to bring about the inclusion of the micro-organisms in the gels which ensures them excellent viability over a period of time.
Now, it became apparent that the micro-organisms enclosed in gels were very sensitive and tolerated storage for a prolonged period of time with difficulty.
In order to overcome this disadvantage of storage, storage processes including a drying have already been suggested.
The commercial preparations of micro-organisms enclosed in dried gels naturally need to be capable of rehydration and maintain excellent viability after rehydration.
Certain solutions have been proposed which permit storage over periods which may attain 6 months to 1 year in a protective packing at a relatively low temperature of 4 to 10°C (see BECKER and RAPOPORT in Advances in Biochemical Engineering and Biotechnology, Volume 1987, pages 128 to 171).
The TATE document EP-A-0 065 376 describes a process for the preparation of enzymes immobilized in a gel which is then dried if appropriate and, after drying, is placed in contact with glycerol (page 10, 2nd paragraph and Claim A placing in contact after drying does not make it possible to preserve the structure of the gel which renders its rehydration difficult, a problem which is resolved by the present invention which will be described below.
The document FR-A-2 519 022 describes a process for the preparation of inocula with long viability and having an improved resistance to temperature which comprises a drying of the microorganisms in accordance with various drying processes. This document also describes in its introduction many documents of the prior art relating to drying of gels enclosing micro-organisms.
According to this document FR-A-2 519 022, at the start a culture of micro-organisms is grown in a standard culture medium for several days.
I I 3 To this culture medium, a gellable polymer, which may be a polysaccharide, xanthan or an alginate, may be added.
After gelation which enables the inclusion of a micro-organism in the culture medium to be carried out, drying is undertaken until an activity of water in the inoculum is produced below its critical value to a value lower than 0.5, this value being maintained during storage (see Claim 1, in particular). Preferably, the activity of water in the inoculum is maintained below 0.3 and preferably even below 0.1 (Claim 2).
It is to be noted that according to this document attention is not paid to the special problem of the rehydration of the dry or appreciably dehydrated gel so as to obtain rehydrated gels having a structure approximately identical with that which they possessed before their dehydration.
exaperlee e Now,.exp4~pr4t has shown that the dehydrated or dried particles obtained by the procedure described in th-is document rehydrate with very great difficulty. In the best cases, in the presence of culture medium, the rehydration remains limited at 20% humidity, the particles always remaining very small, dried up, ungraded with respect to size and very hard.
If a supplementation with hydrophilic substances such as carrageenin or carob seed grain is carried out, this causes the gel to become brittle without improving the rehydration of the gelled polymer, and this is particularly true in the case of the employment of an alginate. In the best cases, viabilities incompatible by some few percent with an industrial use may be obtained.
Hence, it appears necessary to dispose of easily rehydratable gels containing a maximum of viable micro-organisms after rehydration, and to do so even after a long period of storage.
Hence, the aim of the present invention is to resolve the new technical problem consisting of the provision of a solution making it possible to dispose of dried or essentially dehydrated gels, easily rehydratable, containing a maximum of viable micro-organisms after rehydration, and to do this even after a prolonged period of storage.
Another aim of the present invention is to resolve the new technical problem consisting in the provision of a solution making it possible to dispose of gels containing a maximum of viable micro-organisms after rehydration, preserving an essentially unchanged structure of the gel after rehydration, maintaining a good stability of this gel compatible with an industrial use of the rehydrated gel.
These technical problems are resolved simultaneously for the first time by the present invention in an extremely simple manner which can be used on an industrial scale.
Thus, according to a first feature, the present invention provides a process for the preparation of microorganisms enclosed in at least partially dehydrated gels exhibiting an improved viability after rehydration, 15 comprising: a) the inclusion of micro-organisms in a polymer solution capable of being transformed into a gel; 0. b) the gelation of the polymer solution O*S* containing the micro-organisms so as to form a gel 20 enclosing the micro-organisms; and c) the drying of the gel enclosing the microorganisms in order to obtain an at least partially dehydrated gel in which the gels having a high concentration of hydrophilic substaces enclosing the micro-organisms are prepared and then subjected to the said drying step.
By "high concentration" is meant a concentration of hydrophilic substances markedly higher than the concentration of this substance usually used as protective 30 drying agent. Preferably, this concentration of hydrophilic substances is at least twice the usual concentration, even better at least five times the usual concentration.
4a According to another advantageous characteristic of the process according to the invention, the abovezlentioned hydrophilic substance is one of low molecular weight. This hydrophilic substance is preferably selected from among polyols such as sorbitol, inositol, glycerol; the sugars such as sucrose, glucose, fructose. Sucrose constitutes the sugar particularly preferred because it enables particularly unexpected results to be obtained.
According to a particularly advantageous embodiment of the .e S S.at 06 S e
S
S process according to the invention, the concentration of sucrose is at least equal to 500 g/l, and preferably about 1000 g/l of the polymer solution capable of being transformed into a gel.
According to another advantageous characteristic of the process according to the invention, a culture of cells of micro-organisms is grown until the stationary phase is attained, which corresponds in particular in the case of yeasts to a low degree of budding or the initiation of division. Preferably, this low degree of budding is lower than This makes it possible to unexpectedly increase the viability of the micro-organisms, particularly in the case of the yeasts.
In accordance with another advantageous characteristic of the. process according to the invention, a gel is prepared having a double layer structure comprising an internal layer or core of gel containing the cells of the micro-organisms and an external layer or envelope of gel practically devoid of micro-organisms. This gel may exhibit the form either of beads, or fibres, as is well known in the techniques of gelation.
In order to produce a gel having a double layer structure, the previously known processes may be used, such as those described for example in the document JP-A-57-150 385 by preferably using the process described in this document which consists of forming the external layer or envelope with a gellable solution. It is also possible to use the technique described in the document GB-A-i 158 662 or US-A-4 386 895 or US-A-3 396 116, or also EP-A-0 140 336 or US-A-3 015 128 or US-A-3 310 612 or also the techniques of inclusion preparations described in an article by P.G. Krouvel in Biotechnology and bioengineering (1980), volume 22, page 681 or the document MicrocapsulesProcessing and Technology (Asaji Kondo) 1979, pages 62 to 66.
In accordance with a particularly advantageous characteristic of the process according to the invention, the thickness of the external layer or envelope, in the case of beads having a diameter of about 4 mm, is less than 0.8 mm.
According to another advantageous characteristic of the process according to the invention, the above-mentioned high concentration of hydrophilic substances in the gel is obtained by soaking the gel in a solution of hydrophilic substances having the said high concentration up to equilibrium, then by subjecting the gel to the above-mentioned drying step.
In accordance with another advantageous characteristic of the process according to the invention, a drying of the gel enclosing the microorganisms having the above-mentioned high concentration of hydrophilic substances is carried out until an activity of water lower than about 0.5 is obtained.
In accordance with another advantageous characteristic of the process according to the invention, the above-mentioned drying is carried out at a temperature close to 40 0 C and preferably the temperature is increased to attain about 50°C by the end of drying.
In accordance with another advantageous characteristic of the process according to the invention, the above-mentioned drying of the gel enclosing the micro-organisms, containing a high concentration of hydrophilic substances, is carried out in a fluidized bed.
In accordance with another characteristic of the process according to the invention, a drying of the gel containing the micro-organisms containing the above-mentioned high concentration of hydrophilic substances is carried out by means of a lyophilization technique in a vacuum, the temperature of lyophilization is preferably of the order of about -800C 10 0
C.
In accordance with another advantageous characteristic of the process according to the invention, the gel containing the micro-organisms containing the above-mentioned high concentration of hydrophilic substances is preserved at least partially dehydrated in a water vapour-tight packing which is preferably maintained at a relatively low temperature, preferably at about 4 0
C.
In accordance with a preferred characteristic, this storage takes place in a controlled atmosphere very poor in oxygen and enriched in C02.
In accordance with another advantageous characteristic of the process according to the invention, the micro-organisms are yeasts, t I j i" 4J2c' in particular of the genus Saccharomyces and Schizosaccharomyces.
In accordance with a second feature, the present invention also relates to the use of the gels enclosing at least partially dehydrated micro-organisms mentioned above for the preparation of fermented or refermented drinks as well as for the preparation of ethyl alcohol.
The present invention also relates to the gels including the micro-organisms having the above-mentioned high concentration of hydrophilic substances as a novel product, where appropriate in a rehydiated state.
The invention makes it possible to achieve the previously mentioned non-evident, unexpected technical results by resolving the new technical problems previously set out by discovering in an unexpected manner that it was possible to preserve both of the structure of the gels and the viability of the micro-organisms if a high concentration of hydrophilic substances was used. As has been mentioned previously, these hydrophilic substances are preferably selected from among the polyols such as sorbitol, inositol, glycerol; the sugars, in particular sucrose, glucose, fructose; the sugar presently much preferred is sucrose. The high concentration of sucrose is preferably at least 500 g/l, and more preferably about 1000 g/l of the polymer solution capable of being transformed into a gel.
In this way, at least partially dehydrated gels are obtained which can easily be rehydrated even after a prolonged period of storage.
The general conditions of the process for the preparation of the at least partially dehydrated gels including the micro-organisms are the following: a) a culture of the micro-organisms is first grown in a suitable culture medium containing a carbon source, in particular carbohydrates, until the stationary phase is obtained, which corresponds in particular in the case of the yeasts to a low degree of budding or cell division, which is preferably lower than b) An inclusion of the micro-organisms present in the culture medium in an easily gellable or solidifiable polymer is carried out, as is standard.
A
This inclusion may be done by the standard technique of droplet formation so as to produce beads of gel enclosing the micro-organisms.
Preferably, according to the invention, an inclusion with a double layer is carried out so as to produce a protective external layer or envelope of gel practically free of cells of micro-organisms.
c) The gel thus formed, in particular in the form of beads or fibres, is soaked in a solution containing a high concentration of hydrophilic substances, such as previously defined, preferably constituted by a sugar.
In the case of the employment of sucrose, the concentration of hydrophilic substances is preferably at least equal to 500 g/l, even better about 1000 g/l of the polymer solution.
This soaking is carried out until an equilibrium between the solution and the gel is obtained.
d) The drying of the thus soaked gel is then undertaken, after separation of the latter from the solution containing the abovementioned hydrophilic substances by using any previously known suitable drying treatment.
It is possible to use, for example, the technique of the fluidized bed, lyophilization or even a desiccator containing a desiccant.
e) It is then possible to store the at least partially dehydrated gel enclosing a micro-organism under an atmosphere enriched in CO 2 f) The particles can then be rehydrated according to the methodology usually used in the case of the dried yeasts already on the market for the purpose of using them.
Other aims, characteristics and advantages of the invention will become clearly apparent in the light of the explanatory description which follows made with reference to examples of the embodiment below in correlation with the appended figures. These examples are naturally given only as illustrations of the invention and hence should in no way be interpreted as constituting a limitation of the scope of the invention. In the present description and the examples, all of the percentages are given by weight, except where indicated otherwise.
Example I: Preparation of at least partially dehydrated gel according to the invention by using as micro-organism a strain of Saccharomyces cerevisiae.
In this example, a strain of Saccharomyces cerevisiae is used as strain of micro-organisms.
A Malt Wickerham with 10 g/l glucose as hydrocarbon source is used as culture medium.
The yeasts are cultivated in Erlenmeyers on a shaking platform at 28°C and harvesting is done after 32 h of culture at a stage at which the micro-organisms have a low degree of budding, preferably not e:ceeding and a content of the reserve substances glycogen and trehalose here 15 and respectively, by determination according to the techniques described by HERBERT et al. in Methods in Microbiology, Volume 5B, 1971, pages 210 to 344.
In accordance with a variant, it is possible to obtain under the same culture conditions higher amounts of reserve substances, for example about 22% of glycogen and about 13% of trehalose by using the modified Malt Wickerham.
The culture medium is centrifuged so as to separate the micro-organisms which are placed in suspension in an aqueous solution.
This latter is then mixed with an aqueous solution of sodium alginate "CECA SG 1100" so as to obtain a final solution of The mixture is then pumped and dripped into a solution of 0.2M CaC12 at pH 7.
After 45 min of contact, the hardened particles, available in the form of beads of calcium alginate enclosing the cells of micro-organisms, are rinsed with distilled water and exhibit a mean diameter of 2.7 mm. These beads contain about 5.10 cells of micro-organisms/ml of beads if these preparations are destined for a process of secondary fermentation according to the Champagne technique, or 2.108 cells/ml of beads in the case of processes of alcoholic fermentation (reaction in closed vessels or batches for preparation of fermented drinks or production of ethanol).
LUl k 1 In accordance with an essential characteristic of the process according to the invention, after formation of these beads, these beads enclosing the micro-organisms are soaked in a solution containing a high concentration of hydrophilic substances of low molecular weight, constituted here by a solution of sucrose having a concentration of 100 g/100 ml. This immersion of the beads in this sucrose solution is maintained until an equilibrium is obtained so as to give rise to a final concentration of sucrose in the beads equal to about 100 g/100 ml of gel.
Other sugars such as glucose or fructose may also be used instead of sucrose. It is also possible to use hydrophilic substances exhibiting a vitreous structure after dehydration such as sorbitol, glycerol, inositol.
The particles thus impregnated are drained then introduced into a dryer with a fluidized bed, for example of the RETSCH type available from Bioblock Scientifics, presented in the 1988 catalogue on page 152, with programmable air fluxes and temperatures between 900-1800 I/min and 40-120°C, respectively.
The desorption isotherms of the beads without the protective hydrophilic substances according to the invention, and containing sucrose and sorbitol at a concentration of 100 g/100 ml as protective hydrophilic substances according to the invention are reported in figure 1.
The equilibrium of the beads is obtained by desorption under different atmospheres controlled by saturated solutions at 250C.
In figure 2, the drying curve produced at 40°C by using ambient air (relative humidity close to 65%) has been plotted.
In accordance with an advantageous characteristic, an activity of water a w equal to about 0.2 is attained by using drier air and also a higher drying temperature. Experiment has shown that the cells of micro-organisms preserve their viability if the temperature is increased to about 50°C at the end of dehydration.
The viabilities observed as a result of the two dehydration treatments by fluidized bed leading to an activity of water included between 0.3 ad 0.4 are reported in table 1 below.
G-1i
I
11 Table 1: of viabilities observed during two drying treatments at 0 C as a function of the state of the culture Phase of the culture Harvest Before drying After drying Beginning of stationary phase buds 99 99 32 h of culture 3% buds 99 99 The measurements of viability were carried out after rehydration of the beads or particles in a 24 g/l sucrose solution (which is close to the composition of wine) for 1 h at room temperature in a shaken medium.
The beads which were translucent at the beginning become opaque again after rehydration.
After rehydration the final water content is identical with the initial water content.
Redissolution of the beads is effected by the use of a sterile solution of glucose and sodium citrate having the following composition: Pancreatic peptone 1.2 g Glucose 10 g NaC1 10 g Trisodium citrate 20 g Distilled water as required 1000 ml It is observed that the yeasts obtained exhibit a remarkable stability in sealed containers at room temperature since a viability of 86% is obtained after 15 days of storage and after 2 months the I I 12 viability remains higher than Furthermore, it is to be noted that a owering of the storage temperature to 4°C makes it possible to maintain a viability higher than 90% after the same period of storage.
The use in a storage atmosphere based essentially on CO 2 at room temperature also improves survival since after 17 days of storage micro-organisms, in this case yeasts, are obtained exhibiting 94% of activity.
Re-introductions into the wine base in the process are quite satisfactory.
A pressure change curve realised with the aid of dried or dehydrated beads according to the invention includes yeasts which were cultivated until a level of glycogen of about 20% in conformity with this example had been obtained, in comparison with control beads prepared with the same yeasts and not subjected to a drying, and forms the subject of figure It is possible to observe from the curve of pressure changes during the course of the process of secondary fermentation that the beads obtained according to the invention after drying in conformity with the process according to the invention and rehydration have a behaviour essentially similar to control beads not subjected to such a drying.
Example II: Preparation of double layer gel It has been possible to observe that the preparation of the micro-organisms enclosed, for example, in beads according to example I, results in very good survival.
Nonetheless, additional researches have made it possible to establish that the dead micro-organisms were not distributed uniformly in the sphere of the gel, preferably of alginate.
The technique of dissolution previously described makes it possible to demonstrate this distribution.
The scheme of the spatial distribution of the dead cells in the particles of example I, which are monolayers, from the external surface of the particles is represented in figure 3.
91% of the dead cells are distributed in the first 400 microns of a sphere 4 mm in diameter.
The experiments carried out by the inventors have made it possible to demonstrate that it is possible to improve the survival of the micro-organisms appreciably if double layer gels are prepared, i.e. comprising an internal layer or core enclosing the cells of microorganisms and an external layer or envelope of protection practically devoid of cells of micro-organisms. The corresponding technique for the preparation of the double layer gel, in particular in the form of beads, is known in the literature presented at the beginning of the description, in particular from GB-l 158 662 or the document Microcapsules Processing and Technology (Asaji Kondo) 1979, page 62.
For a better understanding, the outline of the principle of a conduit with two concentric tubes is shown in figure 4 as a reminder. This conduit 10 comprises a central axial tube 12 into which the mixture of a gel solution, for example a gel of sodium alginate, containing the cells of micro-organisms in suspension is introduced.
In the external tube 14, concentric with the internal tube 12, the gel solution is introduced alone, for example sodium alginate devoid of cells of micro-organisms so as to form double layer drops in a known manner.
It is preferable according to the invention that the external layer forming the protective envelope has a thickness of about 0.35 to 0.70 mm for a diameter of about 4 mm for the beads or droplets.
As a result of this double layer structure, one is thus led to avoid killing the cells of micro-organisms during the drying carried out as previously described.
Example III: Preparation of gel enclosing Saccharomyces uvarum The procedure indicated in example I is used except that a brewery strain, Saccharomyces uvarum, is employed as strain of microorganisms.
The same results are obtained with a viability higher than The re-introductions into the worts of beer of 12.5% degree
I
14 Plato and 80% of target fermented sugars are quite satisfactory.
Example IV: Preparation of gel enclosing Schizosaccharomyces The procedure indicated in example I is used, except that a standard strain of Schizosaccharomyces is employed as micro-organisms.
This strain is reputed for giving very poor survivals under the conditions of standard dehydration.
Nonetheless, according to the invention, in a totally unexpected manner, results comparable to other strains are obtained with a viability higher than 90% after drying and rehydration.
Example V: Preparation of gel enclosing cells of Saccharomyces cerevisiae by means of dehydration in a controlled atmosphere Beads are prepared as in example I.
The beads are placed in water-tight twist-off containers (750 ml), the humidity of the air of which is controlled by saturated salt solutions or solutions of glycerol.
The activity of the water is checked by refractometry in the case of the glycerol solutions or by means of the NOVASINA EJ 3 apparatus in the case of the salt solutions.
The equilibrium of the beads is obtained by desorption.
After 250 to 300 h of being placed in equilibrium, the viability is measured as previously described.
The results obtained are usually worse than those obtained by a more rapid drying in a fluidized bed.
The best degrees of preservation are obtained with an activity of water less than At an activity of 0.8 or more, the survival of the microorganisms is lower than 5% whereas at an activity of 0.11 it is higher than Example VI: Preparation of gel enclosing cells of Saccharomyces cerevisiae by means of lyophilization.
The preparation of the beads is identical with that of example I preferably using sucrose as protective hydrophilic substance .l at the same concentration.
Under the operating conditions, a freezing temperature of -300C leads to a less good preservation of the structures of the particles than a temperature of -7500 to -80°C with a more rapid rate of cooling.
The frozen particles are placed in a USIFROID SMJ lyophilizer.
The sublimation is conducted in a quite high vacuum ((0.025 mmHg), the reheating is carried out in stages at -40 0 C, 0 0 C and 20 0
C.
The complete cycle takes 24 h to result in an activity of water of 0.4.
The survival levels of the cells of micro-organisms obtained are always higher than 80 0
C.
The conditions of rehydration are identical with those used in example I.

Claims (23)

1. A process for the preparation of micro-organisms enclosed in at least partially dehydrated gels exhibiting an improved viability after rehydration, comprising: a) the inclusion of micro-organisms in a polymer solution capable of being transformed into a gel; b) the gelation of the polymer solution containing the micro-organisms so as to form a gel enclosing the micro-organisms; and c) the drying of the gel enclosing the micro- organisms in order to obtain an at least partially dehydrated gel in which the gels having a high concentration of hydrophilic substances enclosing the micro-organisms are prepared and then subjected to the said drying step.
2. A pr cess according to Claim 1, in which the hydrophil-c su1istance is one of low molecular weight.- S3. A process according to Claim 1 or 2, in which the hydrophilic substance is a polyol or a sugar.
4. A process according to Claim 3, in which the S* polyol is sorbitol, inositol or glycerol. A process according to Claim 3, in which the sugar is sucrose, glucose or fructose. S* 6. A process according to Claim 5, in which the sucrose is used at a concentration at least equal to 500 g/l of polymer solution capable of being transformed into a gel.
7. A process according to Claim 5 in which the sucrose is used at a concentration of a-out 1000 g/l of polymer solution capable of being transformed into a gel.
8. A process according to any one of the Claims 1 to 7, in which a culture of cells of micro-organisms is grown until a stationary phase is attained, which corresponds in particular in the case of the yeasts to a low degree of S -r S 0S S S S. *r i 0 S.. S S Sl~ S...I budding or initiation of division.
9. A process according to Claim 8, in which the degree of budding or initiation of division is less that A process according to any one of Claims 1 to 9, in which a gel having a double layer structure is prepared comprising an internal layer or core of gel containing the cells of micro-organisms and an external layer or envelope of gel substantially devoid of micro-organisms.
11. A process according to Claim 10, in which the thickness of the external layer or envelope, in the case of beads having a diameter of about 4 mm, is less than 0.8 mm.
12. A process according to any one of Claims 1 to 11, in which the high concentration of hydrophilic substances in the gel is obtained by soaking the gel in a solution of the hydrophilic substances having the said high concentration, up to equilibrium, then subjecting the gel to the drying step.
13. A process according to any one of claims 1 to 12, in which drying of the gel enclosing the micro-organism, having the above-mentioned high concentration of hydrophilic substances, is carried out until an activity of water lower than about 0.5 is attained.
14. A process according to any one of claims 1 to 13, in which the drying is performed at a temperature close to 40 0 C and the temperature is increased until about 50 0 C is attained at the end of drying. A process according to any one of Claims 1 to 14, in which the drying of the gel enclosing the micro- organisms and containing a high concentration of hydrophilic substances is performed in a fluidized bed.
16. A process according to any one of Claims 1 to 14, in which drying of the gel containing the micro-organisms, and containing the above-mentioned high concentration of hydrophilic substances, is performed by means of a technique of lyophilization in a vacuum.
17. A process according to Claim 16, in which the temperature of lyophilization is of the order -80 0 C 10 0 C.
18. A process according to any one of Claims 1 to 17, in which the at least partially dehydrated gel containing the micro-organisms, and containing the high concentration of hydrophilic substances, is preserved in a water vapour- tight packing.
19. A process according to Claim 18, in which the water vapour-tight packing is maintained at a relatively low temperature. A process according to Claim 19, in which the relatively low temperature is about 4 0 C.
21. A process according to any one of Claims 18 to in which the preservation takes place in a controlled atmosphere deficient in oxygen and enriched in CO 2
22. A process according to any one of Claims 1 to 21, in which the micro-organisms are yeasts.
23. A process according to Claim 22, in which the yeasts are of the genus Saccharomyces or Schizosaccharomyces.
24. Use of at least partially dehydrated gels containing a high proportion of hydrophilic substances enclosing micro-organisms for preparation of fermented or Srefermented drinks as well as for the preparation of ethyl alcohol, in particular those obtained by the process according to any one of the Claims 1 to 23.
25. Gels having a high concentration of hydrophilic substances enclosing micro-organisms obtained by the process according to any one of Claims 1 to53, where appropriate in a rehydrated state.
26. A process for the preparation of micro-organisms enclosed in at least partially dehydrated gels exhibiting improved viability after rehydration substantially as hereinbefore described with reference to any one of the foregoing examples.
27. A gel having a high concentration of hydrophilic substances enclosing micro-organisms substantially as hereinbefore described with reference to any one of the foregoing examples.
28. A process for the preparation of micro-organisms enclosed in at least partially dehydrated gels exhibiting improved viability after rehydration substantially as hereinbefore described with reference to any one of the accompanying drawings.
29. A gel having a high concentration of hydrophilic substances enclosing micro-organisms substantially as hereinbefore described with reference to any one of the accompanying drawings. DATED this 12th day of July 1993 CHAMPAGNE MOET CHANDON By Their Patent Attorneys: GRIFFITH HACK CO Fellows Institute of Patent Attorneys of Australia o o i C C C
AU39612/89A 1988-07-07 1989-07-06 Process for preparing micro-organisms incorporated within substantially dehydrated gels, gels obtained and their use in the preparation of fermented beverages Ceased AU641360B2 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
FR8809249A FR2633937B1 (en) 1988-07-07 1988-07-07 Process for the preparation of microorganisms included in substantially dehydrate gels, gels obtained and their use for the preparation of fermented beverages
FR8809249 1988-07-07

Publications (2)

Publication Number Publication Date
AU3961289A AU3961289A (en) 1990-02-05
AU641360B2 true AU641360B2 (en) 1993-09-23

Family

ID=9368206

Family Applications (1)

Application Number Title Priority Date Filing Date
AU39612/89A Ceased AU641360B2 (en) 1988-07-07 1989-07-06 Process for preparing micro-organisms incorporated within substantially dehydrated gels, gels obtained and their use in the preparation of fermented beverages

Country Status (7)

Country Link
EP (1) EP0350374B1 (en)
AT (1) AT66689T (en)
AU (1) AU641360B2 (en)
DE (1) DE68900229D1 (en)
DK (1) DK1791A (en)
FR (1) FR2633937B1 (en)
WO (1) WO1990000602A1 (en)

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2673122B1 (en) * 1991-02-25 1994-09-09 Moet & Chandon Ionotropic gel deficient in ionic entity of gelling, process for preparing such gel and use thereof in particular in a process for producing sparkling wine.
IT1258418B (en) * 1992-07-09 1996-02-26 Siapa Spa LYOPHILIZED GRANULES CONTAINING FUNGAL MICROORGANISMS, AND PROCEDURES FOR THEIR PREPARATION.
FR2740473B1 (en) * 1995-10-25 1998-01-16 Coletica Esterified polysaccharide particles and polyamine
DE29610945U1 (en) * 1996-06-24 1996-11-21 Fecken Kirfel Gmbh & Co Maschi Contour cutting machine
FR2812655B1 (en) * 2000-08-04 2003-12-19 Lallemand Sa PREPARATION OF ACCLIMATED MICROORGANISMS IMMOBILIZED IN BALLS, PROCESS FOR THE PRODUCTION THEREOF AND APPLICATION OF SAID PREPARATION TO THE RESUMPTION OF STOPPED FERMENTATIONS
WO2004090128A1 (en) * 2003-04-09 2004-10-21 Proenol Industria Biotecnologica, Lda Method for immobilising microorganisms, related material, and use thereof

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0065376A1 (en) * 1981-05-11 1982-11-24 TATE & LYLE PUBLIC LIMITED COMPANY Immobilized enzymes

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2519022B1 (en) * 1981-12-29 1985-03-15 Rhone Poulenc Sa
FR2586256B1 (en) * 1985-06-21 1987-08-21 Durand Muriel Process for preparing coated live cells for their biotechnological exploitation
FR2600673B1 (en) * 1986-06-26 1989-04-21 Charbonnages Ste Chimique Improved process for microencapsulation of microorganisms in a polysaccharide matrix

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0065376A1 (en) * 1981-05-11 1982-11-24 TATE & LYLE PUBLIC LIMITED COMPANY Immobilized enzymes

Also Published As

Publication number Publication date
EP0350374B1 (en) 1991-08-28
DK1791D0 (en) 1991-01-04
DE68900229D1 (en) 1991-10-02
AU3961289A (en) 1990-02-05
AT66689T (en) 1991-09-15
DK1791A (en) 1991-03-06
EP0350374A1 (en) 1990-01-10
FR2633937B1 (en) 1992-04-03
FR2633937A1 (en) 1990-01-12
WO1990000602A1 (en) 1990-01-25

Similar Documents

Publication Publication Date Title
US6033887A (en) Dehydrated polysaccharide gel containing microorganisms, a sugar and a polyol for producing fermented drinks
Peinado et al. Yeast biocapsules: a new immobilization method and their applications
US5389532A (en) Process of producing a dehydrated polysaccharide gel containing microorganisms for preparing fermented drinks
Silva et al. Malic acid consumption by dry immobilized cells of Schizosaccharomyces pombe
AU641360B2 (en) Process for preparing micro-organisms incorporated within substantially dehydrated gels, gels obtained and their use in the preparation of fermented beverages
CA2405125C (en) Method of producing active dry yeast
Tsakiris et al. Immobilization of yeast on dried raisin berries for use in dry white wine-making
Lee et al. Production of γ-decalactone from ricinoleic acid by immobilized cells of Sporidiobolus salmonicolor
ES2621119B1 (en) Wood shavings with microorganisms, their preparation and use
HU185471B (en) Process for preparing fermented alcoholic products
Gonzalez et al. Production of wine starter cultures
Iconomopoulou et al. Low temperature and ambient temperature wine making using freeze dried immobilized cells on gluten pellets
López-Menchero et al. Effect of calcium alginate coating on the cell retention and fermentation of a fungus-yeast immobilization system
AU2001270696B2 (en) Method for culturing micro-organisms in reducing conditions obtained by a gas stream
Divies et al. Wine production by immobilised cell systems
Hansson et al. Effects of cultivation techniques and media on yields and morphology of the basidiomycete Armillaria mellea
ES2650273T3 (en) Method for alcoholic fermentation of honey must, with high sugar content
US20030186403A1 (en) Preparation of immobilised acclimated micro-organisms, production method and use for preactivating interrupted permentation processes
Lević et al. 18 Immobilised Yeast in Winemaking
CN1186452C (en) Microbial synchronous n-tetradecane fermenting process to produce tetradecadicarboxylic acid
US4397877A (en) Heat treatment of active dried yeast and product thereof
Lević et al. Immobilised Yeast in Winemaking
DD261806A1 (en) Method for the enzymatic manufacture of sect and wine with immobilized microorganisms
RU2239658C1 (en) Method for preparing biocatalyst for producing alcohol-containing sparkling drinks
Gurazi et al. Influence of the medium on the alcoholic fermentation performance of two different immobilization yeast techniques compared to free yeast cell fermentation

Legal Events

Date Code Title Description
MK14 Patent ceased section 143(a) (annual fees not paid) or expired