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

Definitions

• the present invention relates to the field of the production of freeze-dried microorganisms, in particular freeze-dried bacteria, and it is particularly interested in freeze-dried lactic acid bacteria, while endeavoring to propose new operating conditions making it possible to increase the viability of lyophilized microorganisms in during their subsequent conservation.
• all types of microorganisms bacteria, yeasts, molds ...) are a priori freeze-dried, lyophilization is used in particular to keep the strains in the collections of microorganisms. Lyophilization is for example practiced on lactic acid bacteria, probiotic strains, yeasts, and for a wide variety of industrial uses.
• microorganisms are used in particular for the biotransformation of raw materials in the context of:
• lactic acid bacteria in the following, the industrial exploitation of lactic acid bacteria as leavens and probiotic cultures is highly dependent on the preservation technologies used in order to guarantee stable, that is to say, viable and long-lasting cultures. term. Freezing and freeze-drying are commonly used for this purpose, but these techniques provide undesirable side effects which are the denaturation of proteins and the decrease in cell viability.
• Lactobacillus bulgaricus Lb. bulgaricus
• Research conducted with Lactobacillus bulgaricus (Lb. bulgaricus) during drying and storage identified factors such as temperature and water activity of dried powders as critical parameters that could affect survival (see, for example, de Castro et al., 1995 in Applied Microbiology and Biotechnology, 44: 172-176).
• lyophilized strains is made from a parent strain and generally comprises 4 stages: seeding, culture in tank, concentration, conservation. Reference may be made, for example, to the following works: “Freeze Drying and Advanced Food Technology”, Goldblith et al., Académie Press in 1975, or “Treaty de Lyophilisation”, Louis Rey, published by Hermann in 1960. Seeding is carried out from a concentrated culture and in an optimal physiological state. Several techniques can be used among which:
• the actual cultivation stage takes place in the tank, with stirring or not, in a culture medium whose composition is adapted to the specific needs of each microorganism.
• a culture medium whose composition is adapted to the specific needs of each microorganism.
• the composition of the culture medium can be extremely varied, but it is common to mention the presence of one or more of polysaccharides, glycerol, milk, glucose etc. ..
• the semi-continuous or "fed-batch" culture used for example for the production of ferments sensitive to a product of fermentation or for the production of biomass sensitive to inhibition by the fermentation substrate, the culture continues without or with recycling, the latter being used in particular for the production of ferments, molecules of interest, and for the biological purification of wastewater.
• the preservation step can be in liquid form, by freezing, cryo-preservation, lyophilization or drying.
• Protective agents are used to preserve microorganisms from the harmful effects of conservation treatments.
• Lyophilization is known to be a low-temperature dehydration process which consists in eliminating by sublimation most of the water contained in the product after freezing.
• the redox potential (often referred to in the Eh literature) is a physicochemical parameter which, by its nature, is present in all media, provided that they contain at least one molecule that can pass from one molecule to another. an oxidized to reduced state and vice versa. That's why its effect is noticeable on all functions cell. Its action has been shown on various types of microorganisms and especially bacterial strains:
• a gas-bound reductant E allowed to modify the metabolic fluxes in Saccharomyces cerevisiae with an increase of the ratio glycerol / ethanol and the accumulation of the reserve sugars with increase of the survival of the yeasts during the conservation in the liquid state (We refer to the document FR-2,811,331 in the name of the Applicant).
• Eh is already indirectly taken into account through oxygen whose inhibitory effect on lactic acid bacteria is well identified. This effect is due to their inability to synthesize cytochromes and heme-core enzymes.
• each of the constituents of the medium can offer protection, in particular by allowing the accumulation of solutes, the production of exopolysaccharides and the modification.
• membrane lipid profile especially by increasing the ratio of unsaturated fatty acids / saturated fatty acdes.
• Eh in the perspective of a pharmaceutical or veterinary food application, the variation of Eh must involve compounds that do not alter the characteristics of the product and preserving the safety of the products.
• the treatment medium is thus treated with a treatment gas comprising a neutral gas such as nitrogen, argon, helium, carbon dioxide or a mixture of neutral gases or a reducing gas.
• a treatment gas comprising a neutral gas such as nitrogen, argon, helium, carbon dioxide or a mixture of neutral gases or a reducing gas.
• a neutral gas such as nitrogen, argon, helium, carbon dioxide or a mixture of neutral gases or a reducing gas.
• hydrogen such as hydrogen, or a mixture of such neutral and reducing gases
• the treatment that is to say the gas / liquid contact can be carried out according to one of the methods otherwise well known to those skilled in the art, such as bubbling through the medium using a frit , a membrane or a porous, agitation by a hollow shaft turbine, use of a hydro-injector, ... etc ...
• the present invention thus relates to a process for the production of lyophilized microorganisms, in particular freeze-dried bacteria, in particular freeze-dried lactic acid bacteria, of the type in which one of the steps of the manufacturing process is carried out during a seeding of a medium of culture with one or more strains of microorganisms, characterized in that, prior to the seeding step, the treatment of the culture medium with a treatment gas comprising a neutral gas or a reducing gas or a mixture is carried out such gases to obtain a value of the redox potential Eh of the determined medium, which is lower than the value obtained when the medium is in equilibrium with the air.
• a treatment gas comprising a neutral gas or a reducing gas or a mixture
• said value of the desired redox potential is at least 100 mV less than the value obtained when the medium is in equilibrium with the air. said value of the desired redox potential is negative.
• the seeding is carried out indirectly in that the preculture is carried out beforehand, which is subsequently used for said seeding, and that, before seeding the preculture, treatment of the preculture medium with a pre-treatment gas comprising a neutral gas or a reducing gas or a mixture of such gases, to obtain a value of the potential Redox Eh determined pre-culture medium, which is lower than the value obtained when the pre-culture medium is in equilibrium with the air.
• a pre-treatment gas comprising a neutral gas or a reducing gas or a mixture of such gases, to obtain a value of the potential Redox Eh determined pre-culture medium, which is lower than the value obtained when the pre-culture medium is in equilibrium with the air.
• said treatment or pre-treatment gas is hydrogen or comprises hydrogen.
• said treatment or pre-treatment gas is nitrogen or comprises nitrogen.
• said treatment or pre-treatment gas is a mixture of hydrogen and nitrogen.
• said treatment or pre-treatment gas is argon or comprises argon.
• said treatment or pre-treatment gas comprises hydrogen and / or nitrogen and a complementary gas that is acceptable from the point of view of the subsequent use of the lyophilized microorganisms thus manufactured; said treatment or pre-treatment gas; treatment further comprises a complementary gas which is selected from inert gases, especially helium, and from oxygen, carbon dioxide and nitrous oxide and mixtures thereof in all proportions, preferably from carbon dioxide and oxygen and their mixtures.
• said treatment or pre-treatment gas is a mixture of hydrogen and carbon dioxide.
• the lyophilized microorganisms thus obtained have improved properties, especially in terms of resistance of the strain to lyophilization and resistance during its subsequent storage.
• Sterile skimmed milk (4.5 liters) was treated for 1 hour in a modified 5-liter Schott flask by bubbling two different gases at a flow rate of 150 ml / min; a control condition without bubbling was also performed: - Witness (reference);
• the media (sterile skimmed milk) were then inoculated with a probiotic strain Lactobacillus Casei and then placed in an oven at 37 ° C. for 72 hours.

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• 2005
  • 2005-02-22 FR FR0550483A patent/FR2882369B1/fr not_active Expired - Fee Related
• 2006
  • 2006-02-16 US US11/816,628 patent/US20080268524A1/en not_active Abandoned
  • 2006-02-16 WO PCT/FR2006/050140 patent/WO2006090078A1/fr active Application Filing
  • 2006-02-16 CN CNA2006800055243A patent/CN101124317A/zh active Pending
  • 2006-02-16 JP JP2007555672A patent/JP2008530995A/ja not_active Withdrawn
  • 2006-02-16 EP EP06709519A patent/EP1856241A1/fr not_active Withdrawn
  • 2006-02-16 AU AU2006217752A patent/AU2006217752A1/en not_active Abandoned

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