WO2000049133A1

WO2000049133A1 - Method for producing l-sorbose

Info

Publication number: WO2000049133A1
Application number: PCT/EP2000/001370
Authority: WO
Inventors: Anne-Mette Jakobsen; Kirsten Hogh Nielsen
Original assignee: Basf Aktiengesellschaft
Priority date: 1999-02-19
Filing date: 2000-02-18
Publication date: 2000-08-24
Also published as: KR20010102256A; JP2003521232A; DE19907115A1; NO20013995D0; AU2912400A; CN1346401A; IL144651A0; EP1153120A1; NO20013995L; CA2362926A1

Abstract

The invention relates to bacteria of the Gluconobacter oxydans species that are highly tolerant with respect to D-Sorbite, a method for the production thereof and a method for semi-continuous fermentative production of L-Sorbose from D-Sorbite.

Description

Process for the production of L-sorbose

description

The present invention relates to an improved process for the production of L-sorbose, which in particular facilitates the industrial production of this substance, and to special non-recombinant microorganisms which can be used particularly advantageously in the context of this production process.

L-sorbose is the starting product for the production of 2-keto-L-gulonic acid from which L-ascorbic acid is synthesized on an industrial scale. The production of ascorbic acid or the precursors thereof, e.g. L-sorbose, microbiologically by fermentation offers the advantage of producing the desired product in enantiomerically pure form.

However, the fermentative processes used up to now to produce L-sorbose have several disadvantages. A first serious shortcoming of previously known processes is that the starting product, D-sorbitol, could be used in a relatively low concentration, namely in the range of only about 1-15% by weight. The L-sorbose concentration after the end of the fermentation was correspondingly low even when the reaction was complete. A second serious disadvantage was the necessity that the implementation had to be carried out discontinuously, ie in batch mode. This means that a new vaccine culture (inoculum) must first be prepared in several stages for each production batch. This is particularly laborious and time-consuming and represents a serious disadvantage for the large-scale production of L-sorbose. The main reason for these disadvantages is that the microorganisms used for the fermentation, especially bacteria of the Gluconobacter oxydans type, under the conditions of a fermentation on a technical scale, such as high D-sorbitol concentration, inexpensive, inexpensive nutrient medium, pH fluctuations in the range from about 3 to 7, fluctuations in the oxygen supply in the range from 0.1 to 1.5 1 0 ₂ / l medium / h have an extremely limited lifespan. Furthermore, deviations of the reaction conditions from the ideal parameters, especially in large fermenters, are responsible for shortening the lifespan of bacterial culture. Often, a large part of the bacteria used is no longer viable or reproductive at the end of fermentation. In order to create ideal starting conditions for the next reaction batch, ie to be able to provide an inoculum with a high cell density therefore, according to the state of the art, a new rearing of a fresh inoculum is required.

EP-A-0 758 679 describes an L-sorbose-producing G. oxydans strain with the designation G624 (deposit number FERM-BP 4415), which can be cultivated in 20% sorbitol medium. After 4 days of cultivation, this strain converted D-sorbitol into L-sorbose in high yield. This strain is also transformed with an expression construct that carries the coding DNA for the enzymes L-sorbose dehydrogenase and L-sorbosone dehydrogenase. The transformant thus obtained is used to produce 2-keto-L-gulonic acid from D-sorbitol. However, the culture medium used for this fermentation only has a D-sorbitol concentration of 5%. A 5-day cultivation is required to complete the implementation. Overall, it can thus be stated that the bacteria known from EP-A-0 758 679 have neither satisfactory properties with regard to D-sorbitol tolerance nor with regard to L-sorbose synthesis performance. The usability of the microorganisms described therein for the semi-continuous or continuous conversion of D-sorbitol is also not suggested.

The object of the present invention was therefore to create the conditions for a more efficient fermentative production of L-sorbose.

This object was achieved in particular by providing a semi-continuous, fermentative process for the production of L-sorbose from D-sorbitol using microorganisms of the Gluconobacter oxydans species. This task was also solved by providing optimized bacteria of the Gluconobacter oxydans species.

A first object of the present invention thus relates to highly tolerant bacteria of the Gluconobacter oxydans species, which are obtainable by conditioning a low-tolerant G. oxydans strain by cultivating it step by step with increasing D-sorbitol Concentration adapted to an increased D-sorbitol concentration in the culture medium.

A "highly tolerant" G. oxydans strain which is adapted to high D-sorbitol concentrations according to the invention is present when it can be cultivated in media which have an initial D-sorbitol concentration in the range from about 20 to 40% by weight. %, such as, for example, about 25 to 38% by weight or 28 to 35% by weight, cultivation at a D-sorbitol concentration of less than 20% by weight is natural. lent also possible. When cultivated, such a strain should preferably also ferment the D-sorbitol contained in the medium in high, for example 70 to 100%, yield to L-sorbose.

For example, the highly tolerant strains according to the invention are obtainable by conditioning the D-sorbitol concentration in the course of conditioning from initially approximately 5 to 10% by weight to a final concentration in the range from approximately 20 to 40% by weight, e.g. about 25 to 38% by weight or 28 to 35% by weight, gradually increased. Conditioning can be carried out as follows, for example:

A sterile, known per se liquid medium suitable for the cultivation of G. oxydans is prepared, the initial D-sorbitol content of which is adjusted such that after inoculation it has a D-sorbitol content of, for example, about 5 to 15% by weight, such as about 10% by weight. Suitable formulations for culture media are described, for example, in "Industrial Microbiology, Hans-Jürgen Rehm, Springer Verlag Berlin, Heidelberg, New York, 2nd Edition, p. 500. For example, a 10% sorbitol medium (pH 5), the 0 , 5% by weight of yeast extract and optionally 0.1% by weight of glucose were added This medium is inoculated with a freshly prepared inoculum of an unconditioned G. oxydans strain and the inoculated medium is cultured at about 30 to 37 ° C. aerobic conditions and, for example, gentle shaking or stirring. The fermentation is continued until no significant L-sorbose formation can be observed. An aliquot is removed from the fermentation broth and inoculated therewith another sterile culture medium of the second cultivation stage, the D Sorbitol content (after inoculation) is higher than the content in the medium of the previous cultivation stage The ratio of inoculum to culture to be inoculated should be in the range from 1: 5 to 1:20, such as 1: 5 to 1:10. The above procedure is repeated with a gradual increase in the sorbitol concentration until a bacterial culture is obtained which grows in the medium with the desired high initial D-sorbitol concentration. The gradual increase in the D-sorbitol concentration can be selected depending on the response of the parent strain to the increase in concentration. In addition, the increase can take place in the same or different sizes. For example, a gradual increase in the D-sorbitol concentration can take place in the same 0.5% to 3% steps, such as 1% or 2% steps. According to a further embodiment, bacteria of the type G. oxydans are provided which, in addition to an increased D-sorbitol tolerance, also have an essentially constant, ie stable, high L-sorbose synthesis performance. Bacteria with such a property profile can be obtained by repeating a sorbitol-highly tolerant bacterium as described above at an increased sorbitol concentration, for example in an approximately 25 to 35% by weight D-sorbitol medium, until termination of L-sorbose formation.

A satisfactorily "high" L-sorbose synthesis performance is achieved when about 90 to 100 mol%, such as, for example, about 93 to 98 mol%, of the sorbitol used after not more than about 48 hours, in particular after no more than about 36 hours, such as after about 15 to 25 hours or 18 to 22 hours. Since the synthesis performance can be influenced by the culture conditions, the above criteria apply in particular under the following standard conditions: D-sorbitol starting content: 28-30% by weight; pH of the medium: 4.5-5.0; Ventilation 0.5-1.5 1 air / 1 medium / h; Volume ratio of inoculum to culture medium presented about 1: 5 to 1: 6; Standard medium: containing per kg medium

Corn steep liquor, 50% dry substance 4.354 g

Ammonium sulfate 0.495 g diammonium phosphate 0.124 g

Magnesium sulfate, heptahydrate 0.082 g

Calcium carbonate 0.472 g

Antifoam 0.150 g

A satisfactorily "constant" or "stable" L-sorbose synthesis performance is given according to the invention if the cultivation (preferably under the above standard conditions) is achieved by serially inoculating an aliquot of the culture broth onto freshly prepared culture medium, preferably at a volume ratio of inoculum to culture medium provided about 1: 5 to 1: 6, can be repeated several times, and without a significant decrease in the L-sorbose synthesis performance of the production strain used. A fluctuation of the synthesis performance within the ranges defined above for turnover and duration of the implementation is possible. A constant synthesis performance should be repeated after 1 to 5 times or more, e.g. Vaccination 10 to 50 times or more may be observed.

The conditioning methods according to the invention can in principle be carried out with all unconditioned G. oxydans strains. However, the conditioning is preferably carried out using Gluconobacter oxydans NRRL-B72. This strain is in the deposit position (Northern Regional Research Laboratory, USA) is freely available.

The present invention relates in particular in the above manner conditioned bacteria of the species G. oxydans which higt befä- ^~~ are to convert, under aerobic conditions D-sorbitol to L-sorbose and wherein the bacterium is also not in the form of individual, paired or short chains mobile, gram-negative rods are present, good growth in semi-solid sorbitol / glucose / yeast extract agar medium and in liquid sorbitol / corn steep liquor medium at a pH of about 3.5 - 6, a temperature of about 25 - 40 ° C. and a D-sorbitol concentration of up to about 40% by weight, such as, for example, over 20 to 40% by weight, in particular about 25 to 35% by weight.

A particularly preferred G. oxydans strain obtained on the basis of the above-described conditioning process, starting from NRRL-B72 and obtained under the internal name "2B ₃ ", has the following properties:

1. Morphological properties of 2B ₃ :

- Gram staining: negative

- Cell shape: chopsticks

- Cell size: 1 - 2 μm - Appearance of the cells: individually, in pairs or in short chains

- Agility: negative

- Spore formation: negative

- Colonies: a) On meat extract / peptone agar (pH 7.0): after 2 days of incubation, faintly visible, blurred, thin-layered colonies sometimes appear, b) After 2 days of incubation on sorbitol / glucose / yeast extract tagar (pH 5) vaulted, round colonies appear in the shape and size of a pin head. The colonies are whitish yellow and shiny.

2. Physiological properties:

- Growth temperature: T = 25 - 40 ° C, optimum at 32 ° C - pH range: growth at pH 3.5 - 6, optimum at pH 4.5 - 5.0

- aerobic

- Catalase: positive

- Oxidase: negative - Nitrate reduction: negative

- Gelatin liquefaction: negative

- H ₂ S formation: negative - Indole production: negative

- Ethanol oxidation to acetic acid: positive

- Starch: not hydrolyzed

- Glycerin, D-mannitol, sorbitol: used - Lactate oxidation: negative

- Acetate oxidation to C0 ₂ and H ₂ 0: negative

The strain produced thus corresponds to the essential features of G. oxydans according to the information in Bergey's Manual of Systematic Bacteriology, (9th edition), p. 275 ff. With regard to further non-limiting characteristics, reference can be made to the information in this standard set of provisions.

In addition to the bacteria of the species G. oxydans specifically described above, the invention also relates to mutants and variants thereof which have essentially the same properties and are suitable for carrying out the process according to the invention for producing L-sorbose, which is described in more detail in the following sections will be described.

Examples of suitable methods for producing mutants and variants of the specifically described microorganisms according to the invention include, but are not limited to: mutagenesis by irradiation with ultraviolet light or X-rays; treatment with a chemical mutagen such as nitrosoguanidine (N-methyl-N'-nitro-N-nitrosoguanidine), methyl methanesulfonate, nitrogen mustard and the like; Gene integration techniques, and transduction using bacteriophages. These methods are known from the prior art and are described, for example, in: J.H. Miller, Experiments in Molecular Genetics, Cold Spring Harbor Laboratory Press, Cold spring Harbor, New York (1972); J.H. Miller, A Short Course in Bacterial Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1991); M. Singer and P. Berg, Genes & Genomes, University Science Books, Mill Valley, California

(1991); J. Sambrook, EF Fritsch and T. Maniatis, Molecular Cloning; A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989); PB Kaufman et al., Handbook of Molecular and Cellular Methods in Biology and Medicine, CRC Press, Boca Raton Florida (1995); Methods in Plant Molecular Biology and Biotechnology, BR Glick and JE Thompson, ed., CRC Press, Boca Raton, Florida (1993); and PF Smith-Keary, Molecular Genetics of Eschirichia coli, The Guilford Press, New York, NY (1989). The present invention also relates to a process for the semi-continuous, fermentative production of L-sorbose, wherein

a) inoculated with a bacterium of the Gluconobacter oxydans type and contained a first culture medium containing D-sorbitol until the D-sorbitol reaction was essentially complete; b) after the reaction has ended, the L-sorbose formed is isolated from the main amount of the culture broth thus obtained; c) with the remaining amount of the culture broth a second D-

Culture medium containing sorbitol in a weight ratio of 1: 4 to 1: 8 is inoculated and cultivated until the D-sorbitol reaction is essentially complete; and d) after the reaction has ended, the L-sorbose formed is isolated from the culture broth;

and wherein the initial D-sorbitol concentration after inoculation of the culture media is in each case up to about 40% by weight. The inoculum has, for example, a cell density corresponding to 1 to 10 g of biomass (dry substance) per liter of inoculum.

According to a preferred embodiment of the method according to the invention, the fermentation cycle comprising steps a) to c) is repeated as often as required until a sufficient amount of L-sorbose has been formed. An "arbitrary" repetition of fermentation cycles in the context of the present invention means e.g. the 1 to 5 times or more, e.g. repeating the fermentation cycle 10 to 50 times or 50 to 200 times.

It can be seen from this that the method according to the invention offers the particular advantage over the prior art that it does not require a new inoculum for inoculating the production culture medium. Rather, the required inoculum can be branched off directly from the culture broth of the previous fermentation cycle, preferably after the end of the D-sorbitol reaction. Surprisingly, it was also found according to the invention that numerous fermentation cycles can be run through, although the initial D-sorbitol concentration is unusually high, for example in the range 25-35% by weight. It is also surprising that each of the fermentation cycles provides L-sorbose in almost quantitative yield, ie in the range of approximately 95-98 mol%, within a reaction time of approximately 15-25 hours, in particular approximately 20 hours, per cycle. The ratio of inoculum to culture medium chosen according to the invention ensures that the production approach is practically no Phase goes through, but rather contains a bakery culture of high vitality.

The fermentation temperature is preferably approximately 25-40 ° C., in particular approximately 32-36 ° C. A fermentation temperature of about 35 ° C., which lies between the optimum growth of the bacterium (32 ° C.) and the optimum temperature of the enzymatically catalyzed partial oxidation of D-sorbitol to L-sorbose (36 ° C.), is particularly preferred. At temperatures below 25 ° C and above 40 ° C, no significant increase in the number of bacteria can be determined.

While after a drop below 25 ° C with subsequent heating, growth and productivity return to normal values, an increase in temperature to over 40 ° C leads to permanent damage to the growth and productivity of the bacteria.

According to the invention, it is also preferred to carry out the fermentation with the supply of about 0.5-1.5 liters of atmospheric air per liter of medium and per minute. The bacterial growth and the fermentative conversion of D-sorbitol can be further improved in a known manner by enriching the air with oxygen, for example by up to about 20% by volume. The oxygen transition can also be regulated in a manner known per se by varying the pressure. In stirred fermenters, the oxygen transition is influenced in particular by the stirring performance of the stirring device used. Stirring powers of approximately 0.5 to 4 kW / m ^{3 of} medium are preferred. The blade stirrer is an example of a type of stirrer commonly used.

As already mentioned, the bacteria preferred according to the invention have an optimum growth at pH 4.5-5.0. With a pH of over 6 to about 7.5, growth and productivity are reversibly prevented, while the pH in the course of the fermentation also briefly values of 3.5 or less, e.g. can take up to about 3.0 without negatively affecting sorbitol oxidation in the current batch as well as growth and productivity in the next fermentation cycle.

Furthermore, it should preferably be noted that the sorbitol substrate does not have too high a proportion of D-glucose impurities, since this causes an extraordinary drop in pH during the fermentation due to the oxidation of D-glucose to gluconic acid.

The generation time of the microorganisms preferably used according to the invention for fermentation also increases with increasing sorbitol concentration in the medium. For example, the generation time is 3 times longer in a 27% medium than in one 10% medium with otherwise identical fermentation parameters. The generation time increases sharply above a concentration range of about 30-32% by weight of sorbitol. The growth stops at a concentration of about 40% by weight. In a 27% sorbent medium, the bacteria reach optimal bacterial density after inoculation with an inoculum in a ratio of 1: 5.6 (inoculum to culture medium) within about 12 to 18 hours. The oxidation of D-sorbitol to L-sorbose only starts after the culture has reached a certain cell density, such as about 30 to 50% of the final density.

The substrate D-sorbitol used should preferably have no contamination of D-mannitol if L-sorbose is to be isolated in crystalline form. D-mannitol is namely converted enzymatically to D-fructose. The latter inhibits the crystallization process in small amounts.

Another advantage according to the invention can be seen in the fact that the production method according to the invention can be carried out using a comparatively simple, inexpensive nutrient or culture medium. This preferably contains per kg of liquid medium:

a) about 100 - 300 g D-sorbitol b) about 4 - 5 g corn steep liquor (50% dry matter) c) about 0.4 - 0.6 g (NH ₄ ) ₂ S0 d) about 0.1 - 0.15 g (NH ₄ ) ₂ HP0 e) about 0.06 - 0.1 g MgS0 ₄ x 7H ₂ 0 f) about 0.5 - 0.9 g CaC0 ₃ and optionally g) an effective amount of anti-foaming agent.

The culture medium is sterilized in a manner known per se before inoculation. For this purpose, the pH of the medium is set to 5.5, for example with approximately 70-80% acetic acid, before sterilization. During the fermentation, the pH is only controlled by the calcium carbonate added. An additional control, such as by adding sodium hydroxide solution to the ongoing fermentation process, is not necessary. During the initial phase of growth, the pH of the culture is approximately 5.5-6 and during the log phase (logarithmic multiplication of the bacteria) of growth, the pH falls to a range of approximately 4.0-4.5 . In addition to the above-mentioned essential components which are completely sufficient for fermentation per se, further components can be added to the nutrient medium used according to the invention if necessary.

For example, one or more other carbon sources may be included, such as Starch hydrolysates, cellulose hydrolysates or molasses; and alcohols such as glycerin.

One or more nitrogen sources may also be included, e.g. Ammonia, ammonium salts of inorganic or organic acids, such as ammonium chloride, ammonium nitrate, ammonium phosphate, ammonium sulfate and ammonium acetate; Urea; Nitrate and nitrite salts and other nitrogenous materials such as amino acids, meat extract, peptone, fish meal, fish hydrolysates, casein hydrolyzates, soybean hydrolyzates, yeast extract, dried yeast, ethanol yeast distillate, soybean meal, cottonseed meal and the like.

In addition, inorganic salts, such as e.g. Salts of potassium, calcium, sodium, magnesium, manganese, iron, cobalt, zinc, copper and other trace elements as well as phosphoric acid.

Suitable trace elements and growth factors can also be added individually or in combination, e.g. Coenzyme A, pantothenic acid, biotin, thiamine, riboflavin, flavin mononucleotide, flavin adenine dinucleotide and other vitamins, amino acids such as cysteine, sodium thiosulfate, p-aminobenzoic acid, niazinamide and the like. These can be used in pure form or in the form of natural materials which contain these substances.

The preferred fermentation technique used in each case depends primarily on the size of the batch. While an aerobic shake culture is suitable in principle for smaller batches, fermentation under submerged aerobic conditions is preferred for carrying out the process according to the invention on an industrial scale.

The conversion of D-sorbitol is monitored in a conventional manner, for example by HPLC analysis of a sample taken. A suitable HPLC column of the type OA KC 7.8 x 300 mm, from Merck, Mobile Phase 0.05 N sulfuric acid, 0.4 ml / min flow rate is suitable for determining the L-sorbose content. The L-sorbose formed is isolated continuously or batchwise using conventional methods. The resulting culture broth is first concentrated in a conventional manner, if appropriate after separating the cell mass, for example by filtration or centrifugation. Concentration takes place, for example, by evaporation at elevated temperature, such as 50 to 80 ° C, and reduced pressure, such as 0.01 to 0.1 bar. The crystals which precipitate are filtered and, if necessary, recrystallized. Further purification steps, such as solvent extraction, chromatography, precipitation or salting out, can be used individually or in combination if necessary.

A particularly preferred embodiment of the method according to the invention is described in the following experimental section.

Example 1: Preparation of various nutrient media:

a) Corn steep liquor / salt mixture

Corn steep liquor, 50% dry matter 240 g (about 200 ml) ammonium sulfate 22.2 g

Diammonium phosphate 6.6 g magnesium sulfate, heptahydrate 3.6 g

Calcium carbonate 48.6 g

With the exception of calcium carbonate, all components are mixed with one another and the pH is adjusted to about 6 with 25% strength sodium hydroxide solution. Calcium carbonate is then added. The pH is checked again and adjusted to 6.5 if necessary.

b) Sorbitol / corn steep liquor / salt agar

The laboratory cultures of Gluconobacter oxydans are stored in slant agar glasses with this solid medium. The inoculation cultures for the initial inoculation of the fermenter are produced on this solid medium in Roux bottles immediately before inoculation.

Sorbitol syrup, 70% by weight dry matter 75.0 g (about 5% sorbitol) corn steep liquor / salt mixture 7.5 g

Agar agar powder 25.0 g Demineralized water 1 000 ml

Sorbitol syrup, corn steep liquor / salt mixture and water are mixed together, heated to 60 ° C. and filtered. The agar powder is then slowly added to the boiling filtrate. The warm medium is then distributed to the culture vessels (9 ml each 30 ml inclined agar glass and 100 ml each 650 ml Roux bottle). The tubes are then closed and autoclaved at 121 ° C for 20 minutes.

c) Sorbitol / glucose / yeast extract agar (pH 5)

This medium is used to check the purity of the culture during fermentation. The medium is suitable for the growth of G. oxydans and is unsuitable for most of the commonly occurring contaminating microorganisms. G. oxydans forms characteristic curved, round, whitish-yellow, shiny cultures after 1 to 2 days of incubation at 32 ° C.

Sorbitol syrup, 70% by weight dry substance 75.0 g (about 5% sorbitol)

Glucose 1.0 g

Yeast extract 5.0 g agar agar powder 15.0 g

Demineralized water 1 000 ml

Sorbitol syrup, yeast extract and glucose are mixed together and then mixed with water. The pH is adjusted to 5.3-5.4 using 12% by weight acetic acid. The agar powder is slowly added to the boiling mixture. The still warm medium is distributed on glass bottles with screw caps and autoclaved for 20 minutes at 121 ° C. After cooling to about 50 ° C, the medium is distributed to sterile plastic petri dishes.

d) meat extract / peptone agar (pH 7)

This medium is used to check the purity of the culture during fermentation. It is a nutrient medium for most commonly occurring contaminating microorganisms. It is not suitable for the growth of G. oxydans. After 2 days of incubation at 32 ° C, G. oxydans forms faintly visible fuzzy colonies on this medium. No growth is usually observed. Meat extract 3.0 g

Peptone 5.0 g

Agar agar powder 15.0 g

Demineralized water 1 000 ml

The medium was produced in analogy to medium c) above. Only the pH is set to 7.

e) production medium

Recipe for 1,000 kg of a 28% sorbitol medium (density 1,097 kg / m ³ ):

Corn steep liquor, 50% dry substance 4.354 kg

Ammonium sulfate 0.495 kg diammonium phosphate 0.124 kg

Magnesium sulfate, heptahydrate 0.082 kg

Calcium carbonate 0.472 kg

Anti-foaming agent 0.150 kg

Sorbitol syrup (70% dry matter; corresponding to 400 kg sorbitol) is diluted with tap water and mixed with the corn steep liquor. Then add the mineral salts, pre-dissolved or suspended in tap water. Then the pH is adjusted to 5.5 with 70-80% acetic acid. Finally, add the anti-foaming agent. The medium is sterilized in a manner known per se at 141 ° C. for 2 minutes.

Example 2: Preparation of a vaccine culture of G. oxydans

The pre-cultures of G. oxydans are stored in the form of agar cultures (medium b) at 5 ° C. With the help of a sterile inoculation loop, five new agar cultures are prepared from an agar culture by incubation at 32 ° C. for 2 days. The bacteria from eight such agar cultures are used to inoculate the same medium in Roux bottles. For this, bacteria of an agar culture are suspended in 2 x 5 ml of sterile 0.9% saline. The suspension is transferred to a Roux bottle. A total of eight inoculated bottles are incubated at 32 ° C for two days and stored at 5 ° C if necessary. The bacteria grown in two Roux bottles are suspended in 2 x 50 ml of sterile 0.9% saline and transferred to a sterile glass jar. This culture serves as an inoculum for sorbitol fermentation in a 50 m ³ fermenter. The cell density of the inoculum is approximately 10 ⁹ bacteria per ml. Example 3: Fermentation of D-Sorbitol

a) First fermentation step

The 50 m ³ fermenter is filled with 15,000 kg of production medium (compare example 1 medium e)), but the sorbitol concentration is only 15% by weight and the content of corn steep liquor and mineral salts has been doubled). The temperature of the medium is set at 35 ° C. A 200 ml inoculum prepared according to Example 2 is transferred sterile to the fermenter. The fermenter is aerated with 1600 - 1800 m ^{3 of} sterile air per hour. The pressure in the fermenter is about 1.3 atm. The initial pH is about 5.5 - 6.0. The fermentation time in this first step is about 30 to 40 hours. At the end of the fermentation, the pH dropped to about 4.0-4.5. The resulting culture broth is either worked up to isolate the L-sorbose formed or used as an inoculum for the second fermentation stage.

b) Second fermentation step

A second 50 m ³ fermenter is filled with 33,000 kg of sterile production medium (28% by weight sorbitol) and inoculated with 6,400 liters of inoculum (corresponding to 7,000 kg of culture from the first fermentation step). The ratio of inoculum to production medium is 1: 4.7. It is then fermented over a period of about 18 to 22 hours until the sorbitol concentration has dropped to less than 0.1% by weight. The L-sorbose yield is about 8.3 kg / m ³ fermenter volume per hour. Another inoculum of 6,400 liters is branched off from the fermentation broth obtained for the next fermentation cycle. The remaining amount of fermentation broth is processed to isolate L-sorbose.

Example 4: Isolation of L-Sorbose

A fermentation broth obtained by a process according to Example 3 comprises about 25 to 26% by weight of L-sorbose, less than about 0.1% by weight of D-sorbitol, residues from the growth medium (dissolved or suspended) and 1 to 2 g Bacterial mass per liter. The fermentation broth is kept at about 50 ° C to avoid further growth of the bacteria and loss of sorbose. First, the fermentation broth is concentrated in a first concentration step to a sorbose content of approximately 42% by weight at a temperature in the range from 60 to 80 ° C. and a pressure of 0.02 bar in a conventional falling film evaporator. In a second step, further concentration takes place in a conventional long tube evaporator at 57 ° C and a pressure of 0.02 bar. This second evaporation step leads to oversaturation of the concentrated L-sorbose solution. The crystal content is in the range from about 30 to 50% by volume. The crude crystal suspension obtained in this way is then transferred to a conventional sedimentation container. After a while, a very dense crystal suspension forms in its bottom area, which is essentially free of bacterial mass. This is separated from the supernatant, centrifuged, washed with water and dried at about 90 ° C. to a residual moisture content of less than 0.1% by weight.

The mother liquor obtained, the washing liquid and the supernatant from the sedimentation container can then, as described above, be worked up further, if appropriate, after combining with the fermentation broth from another batch.

The total yield, based on the D-sorbitol used, is usually in the range from about 89 to 92 mol% after complete working up. The product produced in this way serves as a starting product for the production of L-ascorbic acid.

Example 5: Conditioning Gluconobacter oxydans

a) A sterile, agar-free liquid medium (850 ml total volume in a 2 l culture vessel) is produced analogously to medium c) above (cf. Example 1) and has a D-sorbitol content of approximately 10% by weight. This medium is inoculated with 150 ml of a freshly prepared inoculum of an unconditioned G. oxydans strain.

The inoculated medium (pH 5) is incubated at 32 ° C under aerobic conditions (atmospheric air) and vigorous shaking (80 rpm). The fermentation is continued until no significant L-sorbose formation can be observed. A volume of 150 ml is withdrawn from the fermentation broth and inoculated therewith another sterile culture medium, the D-sorbitol content of which is 11% by weight. The above procedure is repeated with a gradual (1% increment) increase in sorbitol concentration until a bacterial culture is obtained which grows in about 30% by weight D-sorbitol medium.

The G. oxydans culture obtained in this way, adapted to high sorbitol concentrations, can then be inoculated serially in 30% by weight D-sorbitol medium (150 ml inoculum to 850 ml medium) in order to further optimize the L-sorbose synthesis performance. Optimal synthesis performance is achieved when about 90 to 98 mol% of the sorbitol used has been converted after about 24 hours of cultivation.

Claims

claims

1. D-sorbitol high-tolerance bacterium of the Gluconobacter oxydans species, obtainable by having a low-tolerance

G. oxydans strain is conditioned by gradually adapting it to an increased D-sorbitol concentration in the culture medium by cultivation with increasing D-sorbitol concentration from stage to stage.

2. Bacteria obtainable according to claim 1, characterized in that the D-sorbitol concentration is increased in stages from an initial approximately 5 to 10% by weight to a final concentration of approximately 25 to 38% by weight.

3. Bacteria obtainable according to one of the preceding claims, further characterized by an essentially stable L-sorbose synthesis performance.

4. Bacterium obtainable according to claim 3, obtainable by repeatedly cultivating the sorbitol-tolerant bacterium at an increased sorbitol concentration until the L-sorbose formation has ended.

5. Bacteria obtainable according to one of claims 1 to 4, wherein the conditioning is carried out with Gluconobacter oxydans NRRL-B72.

6. Bacteria obtainable according to one of the preceding claims, characterized in that it a) converts D-sorbitol into L-sorbose under aerobic conditions; b) is in the form of single, pair-wise or short chains of immobile, gram-negative, non-spore-forming rods; and c) good growth on sorbitol / glucose yeast extract agar medium and sorbitol / corn steep liquor medium at a pH of 3.5-6, a temperature of 25-40 ° C and a D-sorbitol concentration of up to about 38% by weight .% shows.

7. A process for the semi-continuous, fermentative production of L-sorbose, characterized in that a) inoculating a first culture medium containing D-sorbitol with a bacterium of the type Gluconobacter oxydans and

5 cultured until the D-sorbitol conversion is substantially completed; b) after the reaction has ended, the L-sorbose formed is isolated from the main amount of the culture broth thus obtained; c) with the remaining amount of the culture broth inoculated and cultivated a second culture medium containing 0 D-sorbitol in a weight ratio of 1: 4 to 1: 8 until the D-sorbitol reaction has essentially ended; and d) after the reaction has ended, the L-sorbose formed is isolated from the culture broth; 5 where the initial D-sorbitol concentration after inoculation of the culture media is in each case up to about 38% by weight.

8. The method according to claim 7, characterized in that the fermentation cycle 0 comprising steps a) to c) is repeated as often as desired.

9. The method according to any one of claims 7 and 8, characterized in that the fermentation temperature is about 25 - 40 ° C. 5

10. The method according to any one of claims 7 to 9, characterized in that the pH of the freshly inoculated culture medium is adjusted to about pH 5-6 and drops to not less than about pH 3.5 during the fermentation. 0

11. The method according to any one of claims 7 to 10, characterized in that one carries out the fermentation with the supply of about 0.5 to 1.5 liters of atmospheric air per liter of medium per minute.

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12. The method according to any one of claims 7 to 11, characterized in that culturing after the inoculation over a period of about 15 to 30 hours.

13. The method according to any one of claims 7 to 12, characterized in that a culture medium is used which comprises per kilogram of medium: a) about 100-300 g of D-sorbitol b) about 4-5 g of corn steep liquor (50% dry matter) 45 c) about 0.4 - 0.6 g (NH ₄ ) ₂ S0 ₄ d) about 0.1 - 0.15 g (NH ₄ ) ₂ HP0 ₄ e) about 0.06 - 0.1 g MgS0 ₄ x 7H ₂ 0 f) about 0.5-0.9 g CaCO ₃ g) an effective amount of anti-foaming agent.

14. The method according to any one of claims 7 to 13, characterized in that one uses a bacterium as defined in one of claims 1 to 6.

15. A process for the preparation of 2-keto-L-gulonic acid, characterized in that a) D-sorbitol is fermentatively converted into L-sorbose using a process according to one of claims 7 to 14 and b) the L prepared in this way -Sorbose in situ or after intermediate isolation in a conventional manner in 2-keto-L-gulonic acid.

16. Use of a bacterium according to any one of claims 1 to 6 for the production of L-ascorbic acid from D-sorbitol.

17. A method for producing D-sorbitol-highly tolerant bacteria, characterized in that a bacterium of the species Gluconobacter oxydans is conditioned as indicated in one of claims 1 to 5.