WO1993003133A1 - Process for the preservation of microorganism culture mediums and packaging utilizing this process - Google Patents

Process for the preservation of microorganism culture mediums and packaging utilizing this process Download PDF

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Publication number
WO1993003133A1
WO1993003133A1 PCT/FR1992/000747 FR9200747W WO9303133A1 WO 1993003133 A1 WO1993003133 A1 WO 1993003133A1 FR 9200747 W FR9200747 W FR 9200747W WO 9303133 A1 WO9303133 A1 WO 9303133A1
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WO
WIPO (PCT)
Prior art keywords
envelope
product
packaging according
culture media
packaging
Prior art date
Application number
PCT/FR1992/000747
Other languages
French (fr)
Inventor
Michel Nadaud
Original Assignee
Michel Nadaud
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Michel Nadaud filed Critical Michel Nadaud
Publication of WO1993003133A1 publication Critical patent/WO1993003133A1/en

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/04—Preserving or maintaining viable microorganisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M99/00—Subject matter not otherwise provided for in other groups of this subclass
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor

Definitions

  • the present invention relates to a new method for improving the conservation of culture media for microorganisms. It also relates to new packaging intended to improve the conservation of said environments.
  • Agri-food or biotechnology medical microbiology laboratories currently use increasingly sophisticated culture media. This is particularly the case for media used as medical or veterinary diagnostic tools.
  • US patent 4262091 describes a method and an apparatus for the preparation of agar media contained in petri dishes in a strict anaerobic atmosphere.
  • the culture media thus prepared are strictly limited to strict anaerobic bacteria and not adaptable to other classes of bacteria.
  • US Pat. No. 3,532,604 describes packaging intended to improve the preservation and transport of biological products contained in supports of different shapes by wrapping them in films of different natures (paper, aluminum, copolymers, polyethylene, etc.) adhering completely to the shape of the wrapped object after achieving a partial vacuum.
  • US Pat. No. 4,798,819 discloses a method of packaging biological culture media where the culture media seeded in polystyrene petri dishes are placed in a sterile individual bag where they are in partial vacuum.
  • international application WO89 / 00189 describes bags intended for the storage, under a controlled atmosphere, of culture media in dishes seeded with bacteria.
  • the atmosphere produced in the bag is intended to improve the nutritional qualities of the culture medium without seeking an improvement in the shelf life of the medium.
  • the applicant has, for its part, carried out persistent research to improve the conservation of the culture medium, in particular of the medium before it is sown. He described in the French patent application FR 2 652060 methods of conditioning ready-to-use culture media allowing their prolonged conservation before use, seeking in particular to avoid their desiccation and their oxidation. He proposed, to do this, to enclose these environments, contained by example in petri dishes, in sealed envelopes made of materials chosen according to their barrier properties both with respect to atmospheric oxygen and of the gases injected into the envelope.
  • the applicant has now found that it was possible to further improve the shelf life of the culture media very much before seeding by keeping them in sealed envelopes inside which at least one product has been introduced before sealing (I ) known in cell biology for its inhibitory properties of peroxides or free radicals within the cell.
  • the present invention relates to a new method for preserving culture media of microorganisms. It also relates to a new packaging allowing the conservation of culture media for microorganisms.
  • the present invention relates to a new method for preserving culture media for microorganisms, characterized in that it consists in enfei— ing said culture media in a sealed envelope containing a product (I ) known in cell biology for its inhibitory effect on peroxides or free radicals within the cell.
  • a product (I ) known in cell biology for its inhibitory effect on peroxides or free radicals within the cell.
  • the invention relates to a process as defined above using as product (I) known in cell biology as an inhibitor of peroxides or free radicals, a product chosen from the mixture of ascorbic acid / tocopherol, the super oxide-dismutase, Glutathione / glutathione peroxidase mixtures, NADPH oxidase. Cytochrome P450, xanthine oxidase, carotenoids, amido-21-steroids.
  • the present invention provides packaging for the preservation of sealed micro-organisms consist of a enve ⁇ Loppe culture media containing at least one container itself containing said culture medium, 'characterized in that that said envelope contains a product known in cell biology to have the effect of eliminating peroxides or free radicals within La cel ⁇ lule.
  • at least one product ( II ) will also be introduced into the envelope, the effect of which is to regulate the level of carbon dioxide or eu.
  • the free radical and / or peroxide inhibitor products and the products intended to regulate the carbon dioxide content are in liquid form and are introduced into the enclosure by permeating a solid support.
  • a solid support By way of example of such solid supports, mention may be made of strips of material of the tissue or paper type or any material of the cellulosic type. Mention will also be made of solid tablets incorporating the products (I) and / or C11).
  • the impregnation of a strip consisting of a cellu ⁇ losic derivative will be carried out, which will be bonded to the inner surface of the envelope.
  • Another way of introducing the products (I) and / or ( II ) is to incorporate them into a tablet which will, for example, be glued to the external face of the lid of the Petri dish.
  • products CI) inhibitors of peroxides or free radi ⁇ cals we can use all those conventionally used for this purpose in the pharmaceutical field when looking for such an effect inside cells.
  • the quantity of products (I) and / or (II) to be introduced inside the envelope will be determined experimentally as a function of the nature of the culture medium and the nature of the gaseous atmosphere contained in the envelope. These experimental methods may consist in particular of a posteriori determinations of the efficiency of the medium for the desired application.
  • the gaseous atmosphere inside the envelope is under a pressure preferably at least equal to the atmospheric pressure. It consists of a gas or a mixture of gases not containing oxygen.
  • the gaseous atmosphere will be composed of a mixture of an inert gas, for example nitrogen or helium, and CO -
  • the proportion of inert gas will advantageously be between 90 and 99% and the CO -, in the proportion of 1 to 9%.
  • the sterile culture medium intended for packaging, will be placed in any type of container conventionally used for the preparation and / or preservation of these media. It may in particular be contained in a Petri dish, made of glass or of polystyrene, in a glass tube ⁇ of polystyrene or of polypropylene, or in any other type of container conventionally used for this purpose.
  • the material constituting the envelope will be * * advantageously chosen from materials combining good mechanical resistance, good insulation with respect to the penetration of oxygen and light, good protection against internal condensation of the water produced by the agar medium. It must also have good weldability properties.
  • ultra-layer complex materials in particular multilayer materials known in food packaging.
  • multilayer materials consisting of three layers: - a layer providing mechanical strength at the same time as protection against light and oxygen, for example a layer aluminum or copolymer of ethylene and vinyl alcohol (EV0H, for example),
  • a layer providing mechanical strength at the same time as protection against light and oxygen for example a layer aluminum or copolymer of ethylene and vinyl alcohol (EV0H, for example)
  • FIGS 1 and 2 which respectively represent a perspective view and a view in longitudinal section of such packaging.
  • Figures 2a and 2b show the magnifications of parts A and B of Figure 2.
  • the culture medium is contained in a Petri dish 2 provided with a cover and placed inside an envelope 1 made of a packaging film.
  • This envelope has two transverse welds 4,
  • FIG. 2 presents a longitudinal section made in the central zone of the same device and FIGS. 2a and 2b are enlarged transverse sections made in A and B in the upper and lower part of the envelope, the latter being in this case formed from three layers 11, 12, 13 respectively made of aluminum, polyethylene and polyester, respectively.
  • the embodiment described above has the advantage of offering a packaging technique which is easily automated and which can be carried out using machines usually used for flo-pack type packaging operations in industry. food.
  • the complex film used to make the packaging can be prepared in reels, the size of which will vary with the type of flow-pack machine and the number and shape of the containers containing the medium to be packaged simultaneously.
  • the internal atmosphere is obtained during the operation of formatting the envelope by the machine thanks to a cannula delivering a gaseous mixture inside the envelope in forma ⁇ tion and until the time of final sealing.
  • the flow rate of the injected gas will take into account the rate of
  • the machine for formatting and welding envelopes make sure that all of the air inside the envelope has been replaced by the gas mixture.
  • the present invention makes it possible to obtain a very markedly increased conservation of the culture media, as is apparent from the examples given without limitation.
  • Example 1 A culture medium is prepared in a conventional manner intended to allow the growth of the bacterium Haemophilus influenzae (designated in this example by strain X) and referenced under the reference ATCC9006 in the American Type of Culture Collection and under the reference IP54 .81 in the collection of the Institut Pasteur.
  • This culture medium contains in particular 0.05% in • volume of a growth factor of strain X consisting of Nicotamide Adenine Diphosphate.
  • the boxes of the first batch (1) are wrapped in welded polyethylene bags and placed at 20 C.
  • the boxes of the second batch (2) are placed in individual sealed envelopes made of a multilayer barrier material and containing a gaseous atmosphere consisting of 98% nitrogen and 2% C0 2 . This batch is stored at 20 ° C.
  • the boxes of the third batch are placed in envelopes identical in all points to those of batch 2 but also containing, stuck on the internal face of the latter, a strip impregnated with a solution containing 2% ascorbic acid,
  • the tests carried out consisted in testing the culture media of the different batches against the strain X at the times: 1 day, 3 weeks, 3 months, 4 months and 6 months.
  • the procedure is as above, but with boxes containing a culture medium intended to inhibit the growth of the Lactobaci L Lus Lactis bacterium referenced under the reference ATCC7830 to the ATCC and under the reference IP53.61 at the Institut Pasteur, ci - 15 below designated by Y and this time by seeding the dishes with a culture broth of the strain Y.
  • the culture medium is a selective medium for Listeria containing, as inhibitor of the Y strain, 0.01% by volume of Trypaflavin.
  • the results of the enumeration in the first quadrant of the colonies of strain Y for the three batches of dishes at the different times are collated in Table II (each figure representing the average obtained for 5 dishes).
  • a control of growth of the germ on medium without inhibitor is practiced in parallel.

Abstract

A process for preserving microorganism culture mediums consisting in enclosing said mediums in a sealed envelope containing a product known in cellular biology for its inhibitory effect on peroxides or free radicals within the cell. The invention also concerns packaging utilizing this process.

Description

Procédé de conservation de milieux de culture de micro-organismes et conditionnement utilisant ce procédé.Method for preserving culture media of microorganisms and conditioning using this method.
La présente invention concerne un nouveau procédé destiné à améliorer la conservation des milieux de culture de micro- organismes. Elle concerne également de nouveaux conditionnements destinés à améliorer la conservation desdits milieux.The present invention relates to a new method for improving the conservation of culture media for microorganisms. It also relates to new packaging intended to improve the conservation of said environments.
Les laboratoires de microbiologie médicale agroalimen¬ taire ou biotechnologique utilisent actuellement des milieux de culture de plus en plus sophistiqués. C'est le cas en particulier des milieux utilisés comme outils de diagnostic médical ou vété- rinai re.Agri-food or biotechnology medical microbiology laboratories currently use increasingly sophisticated culture media. This is particularly the case for media used as medical or veterinary diagnostic tools.
Or, ces milieux, dont la composition est complexe, con¬ tiennent des substances très fragiles au contact de l'air ambiant et de la lumière, même lorsqu'ils sont conservés en ambiance réfri- gérée, en particulier par exemple des vitamines ou des antibio¬ tiques.However, these environments, whose composition is complex, contain very fragile substances in contact with ambient air and light, even when they are stored in a refrigerated environment, in particular for example vitamins or antibiotics.
En outre, la consommation quotidienne, par ces labora¬ toires, de chaque type de produit est trop faible pour autoriser La préparation extemporanée de grandes quantités de ces milieux de culture qui sont périmés avant d'être utilisés. Ceci génère des pertes économiques importantes pour ces Laboratoires.In addition, the daily consumption by these laboratories of each type of product is too low to allow the extemporaneous preparation of large quantities of these culture media which are expired before being used. This generates significant economic losses for these Laboratories.
Du fait de l'augmentation croissante de ces besoins et de la grande variété de ces milieux différents utilisés aujourd'hui, le travail de préparation est trop lourd et fastidieux pour être réalisé quotidiennement convenablement.Due to the increasing increase in these needs and the great variety of these different media used today, the preparation work is too heavy and tedious to be carried out properly daily.
On connaît déjà différents documents décrivant des pro¬ cédés destinés à améliorer la conservation de milieux de culture de micro-organismes avant leur incubation, ou de milieux de culture contenant lesdits micro-organismes. Le brevet US 2 874 091 propose de simplifier Les opéra¬ tions de Lavage et de stérilisation des boîtes de Pétri et décrit en particulier un procédé d'emballage de boîtes en polymères ther¬ moplastiques dans des poches en polyéthylène transparentes, souples, imperméables aux bactéries et permettant d'éviter L'évaporation de L'eau du milieu de culture présent dans La boîte. Dans ce document, l'ambiance intérieure est constituée d'air atmosphérique, ce qui risque d'être nuisible à la bonne conser¬ vation de certains milieux de culture, en particulier Les milieux sensibles à La présence d'oxygène.Various documents are already known which describe processes intended to improve the conservation of culture media of microorganisms before their incubation, or of culture media containing said microorganisms. U.S. Patent 2,874,091 proposes to simplify the washing and sterilization tions opéra¬ petri dishes and particularly describes a process for polymers boxes packaging ther ¬ moplastiques in transparent polyethylene bags, flexible, impermeable to bacteria and making it possible to avoid the evaporation of water from the culture medium present in the box. In this document, the interior atmosphere consists of air atmospheric, which is likely to be detrimental to the good conservation of certain culture media, in particular media sensitive to the presence of oxygen.
Le brevet US 4262091 décrit une méthode et un appareil destinés à La préparation de milieux géloses contenus dans des boîtes de Pétri en atmosphère anaérobie stricte. Les miLieux de culture ainsi préparés sont strictement Limités aux bactéries anaérobies strictes et non adaptables à d'autres classes de bac¬ téries. Le brevet US 3 532604 décrit des emballages destinés à améliorer la conservation et Le transport de produits biologiques contenus dans des supports de différentes formes en Les enveloppant dans des films de différentes natures (papier, aluminium, copoly- mères, poLyéthylène...) adhérant complètement à La forme de L'objet enveloppé après réalisation d'un vide partiel.US patent 4262091 describes a method and an apparatus for the preparation of agar media contained in petri dishes in a strict anaerobic atmosphere. The culture media thus prepared are strictly limited to strict anaerobic bacteria and not adaptable to other classes of bacteria. US Pat. No. 3,532,604 describes packaging intended to improve the preservation and transport of biological products contained in supports of different shapes by wrapping them in films of different natures (paper, aluminum, copolymers, polyethylene, etc.) adhering completely to the shape of the wrapped object after achieving a partial vacuum.
On a également cherché à améliorer la conservation du contenu de bo tes de Pétri ensemencées. Ainsi, le brevet US 4709819 révèle un procédé d'emballage de milieux de cultures biologiques où les milieux de culture ensemencés dans des boîtes de Pétri en polystyrène sont mis sous sachet individuel stérile où elles se trouvent sous vide partiel.Attempts have also been made to improve the conservation of the content of seeded Petri dishes. Thus, US Pat. No. 4,798,819 discloses a method of packaging biological culture media where the culture media seeded in polystyrene petri dishes are placed in a sterile individual bag where they are in partial vacuum.
Par ailleurs, La demande internationale W089/00189 décrit des poches destinées au stockage sous atmosphère contrôlée de milieux de culture en boîtes ensemencés par des bactéries. L'atmosphère réalisée dans La poche est destinée à améliorer Les qualités nutrîtionnelles du milieu de culture sans que soit recherchée une amélioration de la durée de conservation du milieu
Figure imgf000004_0001
Furthermore, international application WO89 / 00189 describes bags intended for the storage, under a controlled atmosphere, of culture media in dishes seeded with bacteria. The atmosphere produced in the bag is intended to improve the nutritional qualities of the culture medium without seeking an improvement in the shelf life of the medium.
Figure imgf000004_0001
Le demandeur a, pour sa part, mené des recherches persistantes pour améliorer la conservation du milieu de culture, en particulier du milieu avant son ensemencement. Il a décrit dans la demande de brevet français FR 2 652060 des procédés de conditionnement de milieux de culture prêts à l'emploi permettant leur conservation prolongée avant utilisation, en cherchant à éviter en particulier leur dessiccation et Leur oxydation. Il a proposé, pour ce faire, d'enfermer ces milieux, contenus par exemple dans des boîtes de Pétri, dans des enveloppes étanches en matériaux choisis en fonction de Leur propriétés de barrière tant vis-à-vis de l'oxygène atmosphérique que des gaz injectés dans l'enveloppe. Le demandeur a maintenant trouvé qu'il était possible d'améliorer encore très sensiblement la durée de conservation des milieux de culture avant leur ensemencement en les conservant dans des enveloppes étanches à l'intérieur desquelles a été introduit avant scellage au moins un produit (I) connu en biologie cellulaire pour ses propriétés inhibitrices de peroxydes ou de radicaux libres au sein de la cellule.The applicant has, for its part, carried out persistent research to improve the conservation of the culture medium, in particular of the medium before it is sown. He described in the French patent application FR 2 652060 methods of conditioning ready-to-use culture media allowing their prolonged conservation before use, seeking in particular to avoid their desiccation and their oxidation. He proposed, to do this, to enclose these environments, contained by example in petri dishes, in sealed envelopes made of materials chosen according to their barrier properties both with respect to atmospheric oxygen and of the gases injected into the envelope. The applicant has now found that it was possible to further improve the shelf life of the culture media very much before seeding by keeping them in sealed envelopes inside which at least one product has been introduced before sealing (I ) known in cell biology for its inhibitory properties of peroxides or free radicals within the cell.
Ainsi, la présente invention concerne un nouveau procédé de conservation de milieux de culture de micro-organismes. Elle concerne également un nouveau conditionnement permettant La conser- vation des milieux de culture de micro-organismes.Thus, the present invention relates to a new method for preserving culture media of microorganisms. It also relates to a new packaging allowing the conservation of culture media for microorganisms.
Selon une caractéristique essentielle, La présente inven¬ tion concerne un nouveau procédé de conservation de milieux de cul¬ ture de micro-organismes caractérisé en ce qu'il consiste à enfei— mer lesdits milieux de culture dans une enveloppe étanche contenant un produit (I) connu en biologie cellulaire pour son effet inhibi¬ teur de peroxydes ou de radicaux libres au sein de la cellule.According to an essential characteristic, the present invention relates to a new method for preserving culture media for microorganisms, characterized in that it consists in enfei— ing said culture media in a sealed envelope containing a product (I ) known in cell biology for its inhibitory effect on peroxides or free radicals within the cell.
Selon une variante de l'invention, elle concerne un pro¬ cédé tel que défini précédemment utilisant comme produit (I) connu en biologie cellulaire comme inhibiteur de peroxydes ou de radicaux libres, un produit choisi parmi le mélange acide ascorbique/ tocophérol, la super oxyde-dismutase, Les mélanges glutathion/ glutathion peroxydase, la NADPH oxydase. Le cytochrome P450, la xanthine oxydase, les caroténoîdes, les amido-21-stéroîdes.According to a variant of the invention, it relates to a process as defined above using as product (I) known in cell biology as an inhibitor of peroxides or free radicals, a product chosen from the mixture of ascorbic acid / tocopherol, the super oxide-dismutase, Glutathione / glutathione peroxidase mixtures, NADPH oxidase. Cytochrome P450, xanthine oxidase, carotenoids, amido-21-steroids.
Selon une autre caractéristique essentielle, la présente invention fournit des conditionnements destinés à la conservation des milieux de culture de micro-organismes constitués d'une enve¬ loppe étanche contenant au moins un récipient contenant lui-même ledit milieu de culture, ' caractérisés en ce que ladite enveloppe renferme un produit connu en biologie cellulaire pour avoir l'effet d'éliminer les peroxydes ou les radicaux libres au sein de La cel¬ lule. Selon un mode de réalisation particulièrement avantageux de L'invention, on introduira en outre dans l'enveloppe au moins un produit (II) dont l'effet est de réguler Le taux d'anhydride carbo¬ ni ue. Selon un mode de réalisation de l'invention particuliè¬ rement avantageux, les produits inhibiteurs de radicaux libres et/ou de peroxydes et les produits destinés à réguler Le taux d'anhydride carbonique sont sous forme liquide et sont introduits dans l'enceinte en en imprégnant un support solide. A titre d'exemple de tels supports solides, on citera des bandes de matériaux de type tissu ou papier ou tout matériau de type cellulosique. On citera également des pastilles solides incor- porant les produits (I) et/ou Cil).According to another essential feature of the present invention provides packaging for the preservation of sealed micro-organisms consist of a enve¬ Loppe culture media containing at least one container itself containing said culture medium, 'characterized in that that said envelope contains a product known in cell biology to have the effect of eliminating peroxides or free radicals within La cel¬ lule. According to a particularly advantageous embodiment of the invention, at least one product ( II ) will also be introduced into the envelope, the effect of which is to regulate the level of carbon dioxide or eu. According to an embodiment of the invention which is particularly advantageous, the free radical and / or peroxide inhibitor products and the products intended to regulate the carbon dioxide content are in liquid form and are introduced into the enclosure by permeating a solid support. By way of example of such solid supports, mention may be made of strips of material of the tissue or paper type or any material of the cellulosic type. Mention will also be made of solid tablets incorporating the products (I) and / or C11).
Dans un mode de réalisation préféré de L'invention, on réalisera l'imprégnation d'une bande constituée d'un dérivé cellu¬ losique, qui sera collée sur la surface intérieure de l'enveloppe. Une autre façon d'introduire Les produits (I) et/ou (II) est de les incorporer dans une pastille qui sera par exemple collée sur la face externe du couvercle de la boîte de Pétri. Comme produits CI) inhibiteurs de peroxydes ou de radi¬ caux libres, on pourra utiliser tous ceux classiquement utilisés à cet effet dans le domaine pharmaceutique lorsque l'on recherche un tel effet à l'intérieur des cellules.In a preferred embodiment of the invention, the impregnation of a strip consisting of a cellu¬ losic derivative will be carried out, which will be bonded to the inner surface of the envelope. Another way of introducing the products (I) and / or ( II ) is to incorporate them into a tablet which will, for example, be glued to the external face of the lid of the Petri dish. As products CI) inhibitors of peroxides or free radi¬ cals, we can use all those conventionally used for this purpose in the pharmaceutical field when looking for such an effect inside cells.
A titre d'exemples non Limitatifs de produits (I), on citera des mélanges acide ascorbîque/tocophérol, La super oxyde- dîsmutase,des mélanges glutathion/glutathion peroxydase, La NADPH oxydase, le cytochrome P450, la xanthîne oxydase, les caroténoîdes, les ubiquînones. Les amido-21-stéroîdes.By way of nonlimiting examples of products (I), mention may be made of ascorbic acid / tocopherol mixtures, super oxide-dimitase, glutathione / glutathione peroxidase mixtures, NADPH oxidase, cytochrome P450, xanthine oxidase, carotenoids, ubiquînones. Amido-21-steroids.
A titre de produits (II) permettant de réguler le taux d'anhydride carbonique dans l'atmosphère à l'intérieur de l'enve- Loppe, on citera tout particulièrement les inhibiteurs de l'anhydrase carbonique, par exemple l'acétazolamide.By way of products (II) making it possible to regulate the level of carbon dioxide in the atmosphere inside the envelope, mention will be made most particularly of carbonic anhydrase inhibitors, for example acetazolamide.
La quantité des produits (I) et/ou (II) à introduire à l'intérieur de l'enveloppe sera déterminée expérimentalement en fonction de la nature du milieu de culture et de la nature de l'atmosphère gazeuse contenue dans l'enveloppe. Ces méthodes expérimentales pourront consister en parti¬ culier en des déterminations a posteriori de l'efficacité du milieu pour l'application désirée.The quantity of products (I) and / or (II) to be introduced inside the envelope will be determined experimentally as a function of the nature of the culture medium and the nature of the gaseous atmosphere contained in the envelope. These experimental methods may consist in particular of a posteriori determinations of the efficiency of the medium for the desired application.
L'atmosphère gazeuse à l'intérieur de l'enveloppe est sous une pression de préférence au moins égale à la pression atmos¬ phérique. Elle est constituée d'un gaz ou d'un mélange de gaz ne contenant pas d'oxygène.The gaseous atmosphere inside the envelope is under a pressure preferably at least equal to the atmospheric pressure. It consists of a gas or a mixture of gases not containing oxygen.
De façon avantageuse, l'atmosphère gazeuse sera composée d'un mélange d'un gaz inerte, par exemple l'azote ou l'hélium, et de CO - La proportion de gaz inerte sera avantageusement comprise entre 90 et 99 % et le C0-, dans la proportion de 1 à 9 %.Advantageously, the gaseous atmosphere will be composed of a mixture of an inert gas, for example nitrogen or helium, and CO - The proportion of inert gas will advantageously be between 90 and 99% and the CO -, in the proportion of 1 to 9%.
Le milieu de culture stérile, destiné à être emballé, sera placé dans tout type de récipient classiquement utilisé pour la préparation et/ou la conservation de ces milieux. Il pourra en particulier être contenu dans une boîte de Pétri, en verre ou en polystyrène, dans un tube de verre^ en polystyrène ou en polypro- pylène, ou dans tout autre type de récipient classiquement utilisé à cet usage.The sterile culture medium, intended for packaging, will be placed in any type of container conventionally used for the preparation and / or preservation of these media. It may in particular be contained in a Petri dish, made of glass or of polystyrene, in a glass tube ^ of polystyrene or of polypropylene, or in any other type of container conventionally used for this purpose.
Le matériau constituant l'enveloppe sera **vantageusement choisi parmi les matériaux associant une bonne résistance méca¬ nique, une bonne isolation vis-à-vis de la pénétration de l'oxygène et de la lumière, une bonne protection contre la condensation interne de l'eau produite par le milieu gélose. Il devra avoir également de bonnes propriétés de soudabilité. On utilisera avanta- geusement, pour réunir toutes ces propriétés, des matériaux com¬ plexes ulticouches, en particulier des matériaux multicouches connus dans l'emballage alimentaire.The material constituting the envelope will be * * advantageously chosen from materials combining good mechanical resistance, good insulation with respect to the penetration of oxygen and light, good protection against internal condensation of the water produced by the agar medium. It must also have good weldability properties. Advantageously, to combine all these properties, ultra-layer complex materials, in particular multilayer materials known in food packaging.
A titre d'exemples de tels matériaux multicouches, on citera des matériaux constitués de trois couches : - une couche apportant la résistance mécanique en même temps que la protection vis-à-vis de La lumière et de l'oxygène, par exemple une couche d'aluminium ou de copoLymère d'éthylène et d'alcool vinylique (EV0H, par exemple),By way of examples of such multilayer materials, mention will be made of materials consisting of three layers: - a layer providing mechanical strength at the same time as protection against light and oxygen, for example a layer aluminum or copolymer of ethylene and vinyl alcohol (EV0H, for example),
- une couche protégeant contre la condensation interne de l'eau contenue dans le milieu de culture, avantageusement constituée de polymères dérivés du polyester ayant subi un traitement anti¬ buée, - une couche présentant de bonnes propriétés de soudabilité et de souplesse, par exemple en poLyéthylène. Un mode de réalisation particulièrement avantageux du conditionnement selon l'invention est représenté aux figures 1 et 2 qui représentent respectivement une vue en perspective et une vue en coupe longitudinale d'un tel conditionnement. Les figures 2a et 2b représentent les grossissements des parties A et B de la figure 2.a layer protecting against internal condensation of the water contained in the culture medium, advantageously consisting of polymers derived from polyester having undergone an anti-fogging treatment, a layer having good weldability and flexibility properties, for example made of polyethylene. A particularly advantageous embodiment of the packaging according to the invention is shown in Figures 1 and 2 which respectively represent a perspective view and a view in longitudinal section of such packaging. Figures 2a and 2b show the magnifications of parts A and B of Figure 2.
En référence à la figure 1, il apparaît que le milieu de culture est contenu dans une bo te de Pétri 2 munie d'un couvercle et placée à l'intérieur d'une enveloppe 1 constituée d'un film d'emballage. Cette enveloppe présente deux soudures transversales 4,Referring to Figure 1, it appears that the culture medium is contained in a Petri dish 2 provided with a cover and placed inside an envelope 1 made of a packaging film. This envelope has two transverse welds 4,
4' et une soudure longitudinale inférieure 5. Cette disposition particulière des soudures permet une ouverture aisée sans recourir à un outil spécial au moment de l'utilisation du milieu de culture.4 'and a lower longitudinal weld 5. This particular arrangement of the welds allows easy opening without using a special tool when using the culture medium.
Sur la face interne de l'envi 'oppe est collée une bande 3 imprégnée du produit (I) inhibiteur de peroxydes ou de radicaux libres et éventuellement d'un produit (II) régulateur du taux deOn the internal surface of the envi ' oppe is stuck a band 3 impregnated with the product (I) inhibitor of peroxides or free radicals and possibly with a product (II) regulating the rate of
C0-, dans L'atmosphère ambiante.C0-, in the ambient atmosphere.
La figure 2 présente une coupe Longitudinale faite dans La zone centrale du même dispositif et les figures 2a et 2b sont des coupes transversales agrandies faites en A et B dans la partie supérieure et inférieure de L'enveLoppe, cette dernière étant dans ce cas formée de trois couches 11, 12, 13 constituées respective¬ ment par exemple d'aluminium, de poLyéthylène et de polyester.FIG. 2 presents a longitudinal section made in the central zone of the same device and FIGS. 2a and 2b are enlarged transverse sections made in A and B in the upper and lower part of the envelope, the latter being in this case formed from three layers 11, 12, 13 respectively made of aluminum, polyethylene and polyester, respectively.
Le mode de réalisation décrit ci-dessus présente l'avan- tage d'offrir une technique de conditionnement facilement automati¬ sable et qui peut être réalisé en utilisant des machines destinées habituellement aux opérations d'emballage de type flo -pack dans L'industrie alimentaire.The embodiment described above has the advantage of offering a packaging technique which is easily automated and which can be carried out using machines usually used for flo-pack type packaging operations in industry. food.
Le film complexe utilisé pour réaliser l'emballage pourra être préparé en bobines dont la lèze variera avec le type de machine flow-pack et le nombre et La forme des récipients contenant le milieu que l'on souhaite emballer simultanément.The complex film used to make the packaging can be prepared in reels, the size of which will vary with the type of flow-pack machine and the number and shape of the containers containing the medium to be packaged simultaneously.
Lorsque le produit (I) inhibiteur de peroxydes ou de radicaux Libres et/ou le produit (II) régulateur du taux d'anhy- dride carbonique sont supportés comme dans l'exemple représenté aux figures 1 et 2 par une bande collée à l'intérieur de L'enveloppe, on pourra avantageusement prévoir de coller cette bande préalable¬ ment sur la face du film complexe destiné à former la face interne de l'enveloppe. On utilisera avantageusement une machine capable de réaliser, selon le mode flow-pack, des emballages en continu à trois soudures, dont deux transversales et une longitudinale. Les deux soudures transversales sont réalisées par un système à mâchoires accompagnantes, de façon à réaliser- une qualité optimale de soudure.When the product (I) inhibitor of peroxides or free radicals and / or the product (II) regulating the carbon dioxide content are supported as in the example represented in FIGS. 1 and 2 by a strip stuck to the inside the envelope, advantageously provision may be made to glue this strip beforehand on the face of the complex film intended to form the internal face of the envelope. Advantageously, a machine capable of producing, according to the flow-pack mode, continuous packaging with three welds, two transverse and one longitudinal, will be used. The two transverse welds are carried out by a system with accompanying jaws, so as to achieve an optimal quality of weld.
L'atmosphère interne est obtenue lors de l'opération de formatage de l'enveloppe par la machine grâce à une canule débitant un mélange gazeux à l'intérieur de l'enveloppe en forma¬ tion et jusqu'au moment du scellage définitif. Le débit du gaz injecté tiendra compte de la cadence deThe internal atmosphere is obtained during the operation of formatting the envelope by the machine thanks to a cannula delivering a gaseous mixture inside the envelope in forma¬ tion and until the time of final sealing. The flow rate of the injected gas will take into account the rate of
La machine à formater et souder les enveloppes. Il faudra s'assurer que la totalité de l'air interne à l'enveloppe a été remplacée par le mélange gazeux.The machine for formatting and welding envelopes. Make sure that all of the air inside the envelope has been replaced by the gas mixture.
La présente invention permet d'obtenir une conservation très nettement accrue des milieux de culture, comme cela ressort des exemples donnés à titre non limitatifs.The present invention makes it possible to obtain a very markedly increased conservation of the culture media, as is apparent from the examples given without limitation.
EXEMPLES Exemple 1 On prépare de façon classique un milieu de culture destiné à permettre la croissance de la bactérie Haemophilus influenzae (désignée dans cet exemple par souche X) et référencée sous la référence ATCC9006 à l'American Type of Culture Collection et sous la référence IP54.81 dans la collection de l'Institut Pasteur. Ce milieu de culture contient en particulier 0,05 % en •volume d'un facteur de croissance de la souche X constitué de Nîcotamide Adénine Diphosphate.EXAMPLES Example 1 A culture medium is prepared in a conventional manner intended to allow the growth of the bacterium Haemophilus influenzae (designated in this example by strain X) and referenced under the reference ATCC9006 in the American Type of Culture Collection and under the reference IP54 .81 in the collection of the Institut Pasteur. This culture medium contains in particular 0.05% in • volume of a growth factor of strain X consisting of Nicotamide Adenine Diphosphate.
On prépare au temps 0, en salle blanche sous flux lami¬ naire stérile, 60 boîtes de Pétri contenant le milieu de culture ci-dessus et on sépare ces boîtes en trois lots :60 Petri dishes containing the above culture medium are prepared at time 0, in a clean room under sterile laminar flow, and these dishes are separated into three batches:
- les boites du premier lot (1) sont enveloppées dans des poches en poLyéthylène soudées et placées à 20 C.- the boxes of the first batch (1) are wrapped in welded polyethylene bags and placed at 20 C.
- Les bo tes du deuxième lot (2) sont placées dans des enveloppes individuelles scellées constituées d'un matériau barrière multicouche et contenant une atmosphère gazeuse constituée de 98 % d'azote et 2 % de C02. Ce lot est conservé à 20°C.- The boxes of the second batch (2) are placed in individual sealed envelopes made of a multilayer barrier material and containing a gaseous atmosphere consisting of 98% nitrogen and 2% C0 2 . This batch is stored at 20 ° C.
- Les bo tes du troisième lot sont placées dans des enveloppes en tout point identiques à celles du lot 2 mais contenant en outre, collée sur la face interne de celle-ci une bande imprégnée d'une solution contenant 2 % d'acide ascorbique,- The boxes of the third batch are placed in envelopes identical in all points to those of batch 2 but also containing, stuck on the internal face of the latter, a strip impregnated with a solution containing 2% ascorbic acid,
(R) 1 % de tocophérol et 0,5 % de Diamox^K Les boîtes de ce Lot sont conservées à 20 C.(R) 1% tocopherol and 0.5% Diamox ^ K The boxes of this Lot are stored at 20 C.
Les essais réalisés ont consisté à tester les milieux de culture des différents lots vis-à-vis de la souche X aux temps : 1 jour, 3 semaines, 3 mois, 4 mois et 6 mois.The tests carried out consisted in testing the culture media of the different batches against the strain X at the times: 1 day, 3 weeks, 3 months, 4 months and 6 months.
Pour ces tests, la valeur d'une anse calibrée d'une sus¬ pension d'une culture de 18 h à 37°C en bouillon approprié de la o souche ajustée à 10 CFU/ml est ensemencée par la technique d'épuisement en quadrants par recouvrement sur Les boîtes des milieux de culture à tester.For these tests, the value of a calibrated loop of a suspension of a culture of 18 h at 37 ° C. in appropriate broth of the o strain adjusted to 10 CFU / ml is sown by the depletion technique in quadrants by overlap on the dishes of the culture media to be tested.
Après une incubation en atmosphère convenable à 37 C pendant 24 h précises, on dénombre les colonies bactériennes pré¬ sentes dans le 4e quadrant.After incubation in a suitable atmosphere at 37 C for 24 h precise, there are paths pré¬ bacterial colonies in the 4 th quadrant.
Des contrôles de croissance sur milieux complets frachement préparés sont pratiqués en parallèle.Growth checks on freshly prepared complete media are carried out in parallel.
Le nombre moyen égal à La moyenne arithmétique du nombre de colonies obtenues sur 5 bo tes est noté et reporté dans le tableau I. Tableau I Dénombrement des colonies de X en fonction du nombre de jours de stockage à 20-C.The average number equal to The arithmetic average of the number of colonies obtained on 5 boxes is noted and reported in Table I. Table I Enumeration of colonies of X as a function of the number of days of storage at 20-C.
0505
Figure imgf000011_0001
Figure imgf000011_0001
-|Q Exemple 2- | Q Example 2
On procède comme précédemment mais avec des boîtes conte¬ nant un milieu de culture destiné à inhiber la croissance de la bactérie Lactobaci L Lus Lactis référencée sous la référence ATCC7830 à l'ATCC et sous la référence IP53.61 à l'Institut Pasteur, ci- 15 dessous désignée par Y et en réalisant cette fois l'ensemencement des boîtes avec un bouillon de culture de la souche Y.The procedure is as above, but with boxes containing a culture medium intended to inhibit the growth of the Lactobaci L Lus Lactis bacterium referenced under the reference ATCC7830 to the ATCC and under the reference IP53.61 at the Institut Pasteur, ci - 15 below designated by Y and this time by seeding the dishes with a culture broth of the strain Y.
Le milieu de culture est un milieu sélectif pour Listéria contenant à titre d'inhibiteur de la souche Y, 0,01 % en volume de Trypaflavine. 20 Les résultats du dénombrement dans le premier quadrant des colonies de la souche Y pour les trois lots de boîtes aux différents temps sont rassemblés dans le tableau II (chaque chiffre représentant la moyenne obtenue pour 5 boîtes).The culture medium is a selective medium for Listeria containing, as inhibitor of the Y strain, 0.01% by volume of Trypaflavin. The results of the enumeration in the first quadrant of the colonies of strain Y for the three batches of dishes at the different times are collated in Table II (each figure representing the average obtained for 5 dishes).
Un contrôle de croissance du germe sur milieu sans 25 inhibiteur est pratiqué en parallèle.A control of growth of the germ on medium without inhibitor is practiced in parallel.
Tableau II Dénombrement des colonies de Y en fonction du temps de stockage à 20°C.Table II Enumeration of the colonies of Y as a function of the storage time at 20 ° C.
3030
Figure imgf000011_0002
Figure imgf000011_0002

Claims

REVENDICATIONS
1. Procédé de conservation des milieux de culture de micro- organismes caractérisé en ce qu'il consiste à enfermer lesdits milieux dans une enveloppe étanche contenant un produit (I) connu en biologie cellulaire pour son effet inhibiteur de peroxydes ou de radicaux libres au sein de la cellule.1. A method for preserving culture media of microorganisms, characterized in that it consists in enclosing said media in a sealed envelope containing a product (I) known in cell biology for its inhibitory effect on peroxides or free radicals within of the cell.
2. Procédé selon la revendication 1, caractérisé en ce que Ledit produit CI) inhibiteur de peroxydes ou de radicaux libres est choisi parmi les mélanges acide ascorbique/tocophérol, la super oxydase-dismutase. Les mélanges glutathion/glutathion peroxydase, la NADPH oxydase, le cytochrome P450, la xanthine oxydase, les caroténoîdes, les ubiquinones, les amido-21-stéroïdes.2. Method according to claim 1, characterized in that Said product CI) inhibitor of peroxides or free radicals is chosen from mixtures of ascorbic acid / tocopherol, super oxidase-dismutase. Glutathione / glutathione peroxidase mixtures, NADPH oxidase, cytochrome P450, xanthine oxidase, carotenoids, ubiquinones, amido-21-steroids.
3. Procédé selon l'une des revendications 1 ou 2, caracté- rîsé en ce que ladite enveloppe renferme en outre un produit (II) destiné à réguler le taux d'anhydride carbonique dans l'atmosphère intérieure à ladite enveloppe.3. Method according to one of claims 1 or 2, characterized in that said envelope also contains a product (II) intended to regulate the level of carbon dioxide in the atmosphere inside said envelope.
4. Conditionnement destiné à La conservation de milieux de culture de micro-organismes constitué d'une enveloppe scellée (1) contenant au moins un récipient C2) contenant lui-même Ledit milieu de culture, caractérisé en ce que ladite enveloppe renferme un pro¬ duit CI) connu en biologie cellulaire pour avoir l'effet d'éliminer les traces de peroxydes ou de radicaux libres au sein des cellules.4. Packaging intended for the conservation of culture media of microorganisms consisting of a sealed envelope (1) containing at least one container C2) itself containing said culture medium, characterized in that said envelope contains a pro¬ duit CI) known in cell biology to have the effect of eliminating traces of peroxides or free radicals within cells.
5. Conditionnement selon la revendication 4, caractérisé en ce que ledit produit (I) est choisi dans la famille des mélanges acide ascorbique/tocophérol, de La superoxydase-dismutase, des mélanges glutathion/glutathion peroxydase, La NADPH oxydase, le cytochrome P450, la xanthine oxydase, Les caroténoîdes, Les ubi¬ quinones. Les amido-21-stéroïdes. 5. Packaging according to claim 4, characterized in that said product (I) is chosen from the family of ascorbic acid / tocopherol mixtures, Superoxidase dismutase, glutathione / glutathione peroxidase mixtures, NADPH oxidase, cytochrome P450, xanthine oxidase, carotenoids, ubi¬ quinones. Amido-21-steroids.
6. Conditionnement selon l'une des revendications 4 ou 5, caractérisé en ce qu'il renferme en outre un produit (II) permet¬ tant deréguLer le taux d'anhydride carbonique, par exemple un inhibiteur de l'anhydrase carbonique. 6. Packaging according to one of claims 4 or 5, characterized in that it also contains a product (II) allows¬ deréguLer the level of carbon dioxide, for example an inhibitor of carbon dioxide.
7. Conditionnement selon l'une des revendications 4 à 6, caractérisé en ce que le produit (I) permettant d'éliminer les radi¬ caux libres ou"les peroxydes et/ou le produit (II) permettant de réguler le taux d'anhydride carbonique sont sous forme liquide et introduits dans l'enveloppe par l'intermédiaire d'un support solide (3) qui en est imprégné.7. Packaging according to one of claims 4 to 6, characterized in that the product (I) for eliminating the free radi¬ cals or " peroxides and / or the product (II) for regulate the carbon dioxide content are in liquid form and introduced into the envelope via a solid support (3) which is impregnated therewith.
8. Conditionnement selon la revendication 7, caractérisé en ce que ledit support solide (3) est constitué par une bande de tissu, papier ou tout type de dérivé de type cellulosique, collée sur la surface interne de ladite enveloppe.8. Packaging according to claim 7, characterized in that said solid support (3) consists of a strip of fabric, paper or any type of derivative of cellulose type, bonded to the internal surface of said envelope.
9. Conditionnement selon l'une des revendications 4 à 8, caractérisé en ce que L'atmosphère gazeuse à l'intérieur de l'enve- loppe est constituée d'un gaz ou d'un mélange de gaz à pression au moins égale à La pression atmosphérique et ne contenant pas d'oxy¬ gène.9. Packaging according to one of claims 4 to 8, characterized in that the gaseous atmosphere inside the envelope consists of a gas or a mixture of gases at a pressure at least equal to Atmospheric pressure and containing no oxygen.
10. Conditionnement selon la revendication 9, caractérisé en ce que ladite atmosphère gazeuse est constituée d'un mélange gazeux contenant 90 à 99 % d'un gaz inerte et 1 à 9 % de C02-10. Packaging according to claim 9, characterized in that said gaseous atmosphere consists of a gas mixture containing 90 to 99% of an inert gas and 1 to 9% of C0 2 -
11. Conditionnement selon l'une des revendications 4 à 10, caractérisé en ce que l'enveloppe (1) est constituée d'un film multi-couche (11, 12, 13).11. Packaging according to one of claims 4 to 10, characterized in that the envelope (1) consists of a multi-layer film (11, 12, 13).
12. Conditionnement selon l'une des revendications 4 à 11, caractérisé en ce que l'enveloppe est scellée par un procédé de type flow-pack.12. Packaging according to one of claims 4 to 11, characterized in that the envelope is sealed by a flow-pack type process.
13. Procédé pour améliorer la conservation des milieux de culture de micro-organismes, caractérisé en ce qu'i.l consiste à enfermer lesdits milieux de cuLture dans des conditionnements selon l'une des revendications 4 à 12. 13. A method for improving the conservation of culture media for microorganisms, characterized in that it consists in enclosing said culture media in packages according to one of claims 4 to 12.
PCT/FR1992/000747 1991-08-01 1992-07-30 Process for the preservation of microorganism culture mediums and packaging utilizing this process WO1993003133A1 (en)

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FR91/09820 1991-08-01

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS62265979A (en) * 1986-05-13 1987-11-18 I S O:Kk Method for cultivating anaerobic mold and tool therefor
EP0246912A2 (en) * 1986-05-23 1987-11-25 Syntex (U.S.A.) Inc. Method and compositions for protecting anerobic microorganisms
US4709819A (en) * 1986-07-23 1987-12-01 Environmental Diagnostics, Inc. Method for preserving plated media and product

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS62265979A (en) * 1986-05-13 1987-11-18 I S O:Kk Method for cultivating anaerobic mold and tool therefor
EP0246912A2 (en) * 1986-05-23 1987-11-25 Syntex (U.S.A.) Inc. Method and compositions for protecting anerobic microorganisms
US4709819A (en) * 1986-07-23 1987-12-01 Environmental Diagnostics, Inc. Method for preserving plated media and product

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Title
BIOTECHNOLOGY AND BIOENGINEERING. vol. XIX, no. 3, 1977, NEW YORK US pages 413 - 424 H.G. SCHLEGEL 'Aeration without Air: Oxygen Supply by Hydrogen Peroxide' *
PATENT ABSTRACTS OF JAPAN vol. 12, no. 155 (C-494)(3002) 12 Mai 1988 & JP,A,62 265 979 ( I S O K.K. ) 18 Novembre 1987 *

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