Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
• • 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
  • C12N1/20—Bacteria; Culture media therefor

Definitions

• the present invention describes a process for obtaining gellan by growing a bacterium belonging to the species Pseudomonas elodea (also called Auromonas elodea) on an appropriate culture medium. This process makes it possible in particular to increase the concentration of gellan in the culture medium while lowering the cost of the production medium.
• Pseudomonas elodea also called Auromonas elodea
• gellan also called heteropolysaccharide S60 by Pseudomonas elodea ATCC 31461 is described mainly in US Pat. a Pseudomonas species: production and basic properties "published in” Applied and Environmental Microbiology ", 1982, vol. 43 n ° 5, pages 1086-1091.
• Gellan is produced by aerobic fermentation on common culture media containing carbon sources, organic and mineral nitrogen sources and mineral salts.
• the nitrogen source for the production of gellan consists of one or more compounds bringing organic nitrogen and possibly a mineral salt, for example NH4NO3.
• These sources of organic nitrogen are compounds rich in proteins or protein hydrolysis residues.
• These sources of organic nitrogen can be yeast extract, malt extract, meat extracts, fish meal, soy flour, cotton meal, maceration liquor corn ("corn steep"), soluble distillation residues, casein hydrolysates, peptones, etc.
• the introduction of such sources of nitrogen into a fermentation medium gives it the name of complex medium.
• a source of mineral nitrogen, in the form of an ammonium salt is sometimes added to the medium.
• These salts can be ammonium sulphate, ammonium chloride or ammonium nitrate.
• the present invention relates to a process for the production of gellan by culture of the micro-organism Pseudomonas elodea ATCC 31461 in a fermentation medium whose nitrogen source consists mainly of nitrate ions.
• the production of gellan that is to say the final concentration of gellan in the fermentation must, is improved.
• the overall gellan productivity that is to say the amount of gellan produced per unit volume of fermentation medium and per unit time of fermentation, as well as the yield of gellan, that is to say the amount of gellan produced divided by the amount of carbonaceous substrate consumed, can also be improved.
• the omission of organic nitrogen sources from the fermentation medium makes it possible to reduce the cost of the medium and therefore to reduce the cost price of gellan.
• the present invention relates to a method for producing gellan in which the culture medium comprises a source of nitrogen mainly consisting of nitrate ions.
• the nitrate ions can come from the group consisting of potassium nitrate, sodium nitrate, dilute nitric acid, and their mixture. Less purified forms, such as those present in certain fertilizers can also be considered.
• the nitrogen source is generally incorporated all at once in the fermentation medium, before sowing.
• the nitrogen concentration, counted in nitrogen N can be between 0.05 and 1 g / l in the culture medium, and preferably between 0.2 and 0.8 g / l.
• the nitrogen source may not contain a significant amount of organic nitrogen or ammonium ions.
• the invention also relates to a culture medium for producing gellan.
• the culture medium comprises a source of nitrogen mainly constituted by nitrate ions.
• Gellan also called heteropolysaccharide S60
• Pseudomonas elodea ATCC 31461 is produced by culturing the microorganism Pseudomonas elodea ATCC 31461 on media containing sources of carbon, nitrogen and mineral salts.
• the carbonaceous substrate preferably contains carbohydrates such as glucose, fructose, maltose, sucrose, xylose or mamitol
• the concentration of carbohydrate in the medium depends in part on the concentration of the other constituents of the medium, in particular the nitrogen concentration.
• the initial concentration of carbohydrate must be chosen so as to minimize as much as possible the residual concentration (that is to say at the end of fermentation) of carbohydrate.
• the initial carbohydrate concentration generally varies from 10 to 60 g / l, preferably from 20 to 40 g / l.
• Example 1 A typical composition of the mixture of these salts is given in Example 1. This composition is given by way of illustration and is not limiting.
• the fermentation according to the present invention is carried out at temperatures between 25 and 35 ° C, preferably between 28 and 32 ° C.
• the pH of the medium is regulated to a value between 6 and 8 throughout the fermentation.
• the production reactor is agitated using agitation modules ensuring good transfer within the reactor.
• the medium must be aerated, the aeration rate being between 0.2 and 2 wm (volume of air per volume of liquid and per minute).
• Fermentation takes place in batches.
• the reactor contains all the ingredients of the culture medium, then is sterilized and, once returned to the fermentation temperature, is then seeded with an active culture.
• the seeding rate that is to say the ratio of the volumes of seeding and medium after seeding, is between 0.1 and 10%.
• Sterilizable probes for recording and regulating parameters such as pH, temperature, dissolved oxygen level, equip the reactor. It is also possible to know the oxygen and carbon dioxide levels in the outlet gases using analyzers placed on the outlet of the reactor gases.
• the culture can be stopped.
• the main criterion for stopping fermentation is low glucose consumption.
• the fermentation must contains raw gellan. This can be recovered by precipitation with an appropriate solvent, most often isopropanol. The recovered precipitate can be dried.
• the total dry matter contains both so-called native gellan and cells from the culture of Pseudomonas elodea. This method of precipitation with isopropanol followed by drying is the conventional method for determining the concentration of crude gellan in the fermentation must.
• the Pseudomonas elodea ATCC 31461 strain is stored in tubes at -22 ° C on a complex agar medium (Nutrient Agar, Difco).
• a tube is returned to room temperature for a few hours and a sample of approximately one ose is taken on the agar surface and spread on a Petri dish containing agar medium (Nutrient Agar , Difco). After 48 hours of incubation at 30 ° C., a development of yellow colonies is observed on the surface of the agar medium. An isolated colony is removed and suspended in 20 ml of liquid medium (Nutrient Broth, Difco) in Erlenmeyer flasks.
• vials are stirred for about 30 hours at 30 ° C.
• the entire contents of a vial, or 20 ml are used to inoculate 180 ml of preculture medium placed in a Fernbach flask.
• the composition of this preculture medium is given in Table 1. All of these transfer operations are carried out under sterile conditions and the media and containers have previously been sterilized under conditions usual for those skilled in the art.
• the Fernbach flasks are stirred for about 40 hours at 30 ° C. After incubation, approximately 180 ml of the contents of a Fernbach flask are used to seed 1.82 liters of production medium contained in a fermenter.
• the composition of the production medium is indicated in Table 1 and corresponds to the optimal composition given in the prior art.
• the 20 ml of remaining medium are used to carry out routine analyzes and to verify the good progress of the preculture.
• the fermenter used is equipped with a multi-hole aerator, a temperature control, a pH probe connected to a regulator controlling the injection of KOH 1N or H3PO4 1 N, a dissolved oxygen probe, a stirring system consisting of an axis provided with two centripetal turbines and driven by a stirring motor.
• the temperature of the medium is regulated at 30 ° C., the pH at 6.5, the air flow rate is 2 wm and the stirring speed is 900 revolutions / minute.
• the main parameters determined during fermentation are the concentrations of glucose, ammonium and nitrate ions, crude gellan and the optical density of the medium read at 620 nm.
• Table 1 Composition of preculture and fermentation media
• the salt solution contains 1.8 g / I MnCl2 ( 4H 2 0
• the glucose concentration is determined using an automatic analyzer (Glucostat, Beckmann) by an enzymatic method with glucose oxidase.
• concentrations of ammonium ions and nitrates are made using Boehringer enzyme kits (respectively referenced 1112732 and 905658).
• the concentration of crude gellan is determined on the fermentation must by precipitation of one volume of medium with two volumes of isopropanol. The precipitation is carried out hot with vigorous stirring.
• the precipitate is recovered by filtration on a glass microfiber filter, with a porosity of 1.2 ⁇ m, previously dried and tared.
• the precipitate is washed with isopropanol.
• the precipitate is then dried and weighed in an infrared desiccator mounted on a balance. This gives the concentration of crude gellan, that is to say the polysaccharide and cell debris.
• the concentration of native gellan thus determined is 6.3 g / l.
• the native gellan productivity is 0.14 gHh- 1 .
• the yield of native gellan relative to the glucose consumed is 0.24.
• Example 2 Preparation on media without organic nitrogen. Three other fermentations (2, 3, 4) were carried out under conditions strictly identical to those described in Example 1 with the exception of the source of nitrogen present in the production medium.
• Fermentations 5, 6, 7 and 8 are carried out under conditions identical to those of fermentation 4, that is to say with KNO3 as the only source of nitrogen in the fermentation medium, the initial concentration of KNO3 being variable.
• the initial KNO3 concentrations as well as the native gellan concentrations obtained and the productivities and yields are summarized in Table 4.
• the results of fermentation 4 are also given in Table 4.

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• 1994
  • 1994-12-26 FR FR9415742A patent/FR2728586A1/fr active Granted
• 1995
  • 1995-12-20 WO PCT/FR1995/001702 patent/WO1996020283A1/fr active Search and Examination

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