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
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
  • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
  • C12P7/18—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic polyhydric

Definitions

• composition of microorganisms and process for the production of xylitol The present application relates to a composition containing a combination of microorganisms for the production of xylitol.
• It also relates to a process for producing xylitol.
• Xylitol is a polyol with five carbon atoms which has a sweetening power close to that of sucrose. Due to its physical properties ⁇ particular chemical, xylitol is used as a substitute sucrose or other sweetening molecules in food to targeted products, pharmaceutical and medical. Indeed, xylitol has a high stability and is inert towards the Maillard reaction. It is not fermentable by many microorganisms and in particular bacteria from dental plaque. Its assimilation is insulin-dependent, which allows its use by diabetics.
• xylitol is naturally present in many fruits and vegetables, but in too small quantities to be extracted profitably.
• xylitol is therefore obtained by chemical reduction of xylose, a monosaccharide with five carbon atoms and major sugar derived from the hemicellulosic fraction of lignocellulosic materials in which it is present in the form of xylans.
• This process for the chemical production of xylitol comprises the following stages:
• the chemical production of xylitol is characterized by a high number of stages, on the one hand that the raw material used is a very complex material from a composition and structure point of view and on the other hand the chemical reduction is non-selective vis-à-vis the substrate, which is composed of a mixture of sugars, mainly comprising xylose.
• This non-specific reduction of xylose leads to the development in the chemical process of heavy separation and purification techniques (chromatography in particular), coupled with recycling operations.
• Candida tropicalis HXP2 (mutant): production of 40 g / 1 of xylitol with a yield of 0.78 g / g and a productivity of 1.6 g / l / h (Gong et al., 1981 Biotechnology Letters, 3 (3), 130-135,).
• Candida tropicalis production of 17 g / 1 of xylitol with a yield of 0.57 g / g and a productivity of 0.35 g / 1.h (Barbosa et al., 1988, The Journal of Industrial Microbiology, 3, 241-251),
• Pachysolen tannophilus production of 4.7 g / 1 of xylitol with a yield of 0.23 g / g and a productivity of 0.1 g / 1.h (Delgenes, 1989, Production ethanol from sugars derived from hemicelluloses by Pichia stipitis. Thesis of Jardin II),
• Candida pelliculosa production of 7.5 g / 1 of xylitol with a yield close to 1 g / g and a productivity of 0.31 g / 1.h (Kitpreechavanich et al., 1984, Biotechnology Letters, 6 (10) , 651-656),
• Candida sp . production of 180 g / 1 of xylitol with a yield of 0.83 g / g and a productivity of 1.6 g / 1.h (Chen and Gong, 1985, Journal of Food Science, 50, 226 -228),
• sorbitol is very difficult to separate from xylitol by chromatographic fractionation.
• the absence of production of this polyol from glucose in the biological process is an advantage.
• L-arabinose is generally converted into arabinitol by yeasts that produce xylitol.
• Candida guilliermondii cultivated on arabinose in microaerobiosis shows a yield of 0.17 g / g (Meyrial et al., 1991).
• Debaryomyces hansenii cultivated under microaerobic conditions, a conversion yield from arabinose to arabinitol close to 100% is obtained (deduced from the results mentioned in WO 91.10.740).
• the applicant therefore set out to implement a microbiological process making it possible to obtain xylitol substantially devoid of arabinitol.
• the subject of the present invention is a composition containing in combination, for simultaneous or separate use over time, at least one first microorganism producing xylitol and at least one second microorganism converting arabinose into metabolites different from arabinitol and not consuming no xylose or xylitol.
• the first microorganism. that is to say that producing xylitol, is a yeast, advantageously of the genus Candida or of the genus Debaryomyces.
• a yeast can be Candida guilliermondii or Candida parapsilosis which constitute the species whose use is preferred in the invention.
• any other yeast having a power to convert xylose to xylitol may be suitable.
• examples of such strains include Candida tropicalis, Candida bo ⁇ dinii, Candida pelliculosa and Debaryomyces hansenii.
• bacteria can be used, in particular of the genus Pediococcus or Propionibacterium or of the genus Lactobacillus, preferably chosen from the species Lactobacillus brevis or Lactobacillus reuteri. But it should be understood that any other microorganism can be suitable as long as it satisfies the following conditions: * inability to metabolize xylose and xylitol,
• the bacterial strain is cultivated on the one hand on a complete liquid medium
• the carbon source is xylose and on the other hand on a complete liquid medium whose carbon source is arabinose.
• HPLC liquid chromatography
• a count under the microscope, after a few days of incubation makes it possible to establish, compared to a count at the initial instant that there has been a multiplication of the two associated strains. All of the above-mentioned experiments were carried out in the laboratory.
• Another possible approach for establishing the criteria required for the bacteria is to carry out the tests on solid medium in petri dishes and to establish the appearance (consumption of arabinose) or not (non consumption of xylose or xylitol) of the bacterial colonies after a few days of incubation.
• arabinosis it is not possible with this approach to easily know whether or not there has been production of arabinitol.
• the invention further relates to a process for producing xylitol, substantially without accumulation of arabinitol, in which the composition of microorganisms described above is cultured in a medium containing at least nutrients and sugars. hemicellulosic origin, in conditions allowing the development of microorganisms.
• nutrients is meant the elements necessary for the growth and metabolic activities of microorganisms, for example yeast extract, peptone and one or more mineral salts.
• mixture of sugars of hemicellulosic origin is meant all the substrates containing xylose and arabinose as major sugars. Such a substrate may be present in the form of a hemicellulose hydrolyzate of plant biomasses.
• Sugars of hemicellulose origin are mainly xylose but may also be other sugars characteristic of hemicelluloses, such as arabinose, glucose, mannose or galactose. It should be noted that they can be replaced, at least partially, by sugars of synthetic origin.
• the present invention therefore makes it possible to solve a problem which has long been posed to a person skilled in the art. It therefore constitutes significant technical progress.
• xylitol has particularly interesting applications in human health and nutrition, as indicated above.
• the method is implemented in a liquid culture medium.
• Partial oxygenation is understood to mean a process in which oxygen is not supplied during part of the process, for example at the start of the process.
• the aeration rate must be sufficient to allow the fermentation of sugars, and in particular xylose, but not excessive, which would decrease the yield of xylitol as a result of increased cell production.
• the optimum value for air flow can be determined by simple tests.
• the oxygen transfer rate it is, for example, from 0.0001 to 500 and preferably between 0.001 and 100 millimoles of oxygen per liter of fermentation medium and per hour. To measure it, it suffices to differentiate between the oxygen introduced into the fermenter and the oxygen leaving the latter, therefore to make the oxygen balance, and to report the result to the volume unit of fermentation medium. and by the hour unit.
• the temperature for carrying out the process is between 15 and 45 "C and preferably between 25 and 35 ° C.
• the pH of the culture medium is, for its part, preferably between 3 and 8 and even more preferably between 5 and 7.
• the culture is generally carried out during 20 to 200 hours in order to obtain an optimal concentration of xylitol.
• the yeast strains producing xylitol are:
• Microbial strains with the inability to metabolize xylose and xylitol and the ability to convert arabinose to metabolites other than arabinitol are Lactobacillus brevis NRRL 3065 and Lactobacillus reuteri NRRL 14171 obtained from the Northern Regional Research Center (Peoria, USA ). These bacterial strains convert arabinose into organic acids (lactic acid, acetic acid). The strains are maintained on agar nutrient media at 4 * C.
• the nutrient medium for yeasts contains 10 g / l of glucose, 5 g / l of pancreatic peptone, 3 g / l of yeast extract, 3 g / l malt extract and 20 g / 1 agar.
• the nutrient medium for bacteria is MRS medium (De Man et al., 1960, J. of Appl. Bact., 23, 130-133) supplemented with 20 g / l of agar.
• Liquid media for the propagation of microorganisms contain:
• the liquid medium comprising 5 g / l of yeast extract, 3 g / l of Yeast Nitrogen Base, 5 g / l of meat peptone-casein, 1.2 g / is introduced into an aerated fermenter.
• the carbon source is a mixture comprising approximately 16.6% glucose, 66.7% xylose and 16.7% arabinose; these proportions simulating a hydrolyzate of hemicelluloses from plant biomasses. Different concentrations of total sugars have been tested: approximately 60 g / 1, 120 g / 1 and 180 g / 1. Yeast and bacteria inocula
• the fermentation is conducted at a pH and a temperature controlled at 6 and 30 ° C respectively.
• the fermenter used has a capacity of 2 liters.
• Xylose, glucose, arabinose, arabinitol and xylitol are quantified by high performance liquid chromatography.
• the total cell development, expressed in g / 1 (dry weight) was measured by gravimetry.
• the duration of the fermentation which is generally between 30 and 200 h, depends on the rate of inoculation of the initial concentration of sugars and the speed of oxygen transfer, the end of the fermentation can be determined by a person skilled in the art by means of regular samples of culture samples.
• P 1F final concentration of xylitol (g / 1)
• P ⁇ Q initial concentration of xylitol (g / 1)
• X 0 initial concentration of xylose (g / 1)
• Xp final concentration of xylose (g / 1) * Yp 2 S yield of arabinitol (g of arabinitol produced per g of arabinose consumed)
• a 0 initial concentration of arabinose (g / 1)
• a F final concentration of arabinose (g / 1) * Purity T in xylitol (%).
• This procedure consists of initial inoculation of the complete liquid medium containing the hemicellulosic sugars as a carbon source, with the bacterial leaven, then when the total consumption of arabinose is obtained, the medium is inoculated with the yeast leaven in order to produce xylitol from xylose:
• anaerobic conditions or microaerobic conditions
• pH regulation at 6
• temperature at 30 ° C, preferably,
• the experiment was carried out in accordance with the general protocol with an aeration flow of 0.005 Volume / Volume per Minute (VVM) and an agitation speed of 800 Rotations Per Minute (RPM) corresponding to an oxygen transfer speed of 1 , 7 ⁇ unole / lh
• VVM Volume / Volume per Minute
• RPM Rotations Per Minute
• the initial sugar concentrations are as follows: xylose: 43.6 g / 1 arabinose: 10 g / 1 glucose: 10 g / 1.
• the process of the invention made it possible to obtain zero production of 1 arabinitol while retaining good parameters for converting xylose to xylitol.
• Analysis of the samples shows that arabinose is converted by Lactobacillus brevis to lactic acid and acetic acid.
• Fermentation is carried out on a mixture of sugars containing (experimental values) xylose (41 g / 1), arabinose (9.7 g / 1) and glucose (11 g / 1) and under the same conditions than in Example 1 except that the bacterium Lactobacillus brevis NRRL 3065 is replaced by the bacterium Lactobacillus reuterii NRRL 14171.
• the fermentation parameters and results are presented in Table 3.
• Lactobacillus reuterii Lactobacillus reuterii.
• Lactobacillus reuterii Parameters Values arabinose conversion rate (%) 100 T (%) 99.5
• the fermentation parameters are presented in Table 4.
• EXAMPLE 6 Fermentation of a hemicellulosic hydrolyzate of wheat straw obtained by acid hydrolysis followed by filtration.
• the hydrolyzate comprising 39 g / l of xylose; 5.9 g / l of glucose and 7.9 g / l of arabinose constitute the carbon source of the fermentation medium.
• the experiment was carried out in accordance with the general protocol using Candida guilliermondii NRC 5578 associated with Lactobacillus reuteri NRRL 14171 and an oxygen transfer rate of 3.9 mmol / lh
• the parameters and the fermentation results are presented in table 8. TABLE 8

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• 1994
  • 1994-05-27 FR FR9406487A patent/FR2720406B1/fr not_active Expired - Fee Related
• 1995
  • 1995-05-24 EP EP95920984A patent/EP0760862A1/de not_active Withdrawn
  • 1995-05-24 AU AU26208/95A patent/AU2620895A/en not_active Abandoned
  • 1995-05-24 WO PCT/FR1995/000681 patent/WO1995033063A1/fr not_active Ceased

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