DE2203467C - Fermentative process for the manufacture of D mannitol - Google Patents

Description

and the hydrocarbon. The best way to achieve this contact is by submerged aerobic activity Fermentation with rapid stirring of the mixture while passing air, e.g. B. through pearls.

During the fermentation, the usual time and temperature conditions known for the cultivation of yeasts, ie about 20 to 34 ° C. for about 4 to 9 days, can be used. In the fermentation process according to the invention, the last production stage is carried out ^{for about 8 to 9 days at 24 to 25 ° C. with stirring.}

The inoculum for the actual fermentation is obtained in several stages. A standard vaccine is prepared from an aqueous suspension of cells from an inclined culture medium of Candida lipolytica ATCC No. 20297, the for the imposition of an aqueous. Glycerin, beef extract, yeast extract and peptone-containing nutrient medium is used. After this nutrient medium has been ^{incubated for about 20 hours at 28 °} C. with stirring, the standard vaccine obtained is used for inoculating Erlenmeyer flasks which contain an aqueous nutrient medium and an assimilable hydrocarbon, e.g. B. n-hexadecane. After about 48 hours of incubation at 24 to 28 ° C., parts of the culture obtained are used for inoculating Fernbach flasks which contain an aqueous nutrient medium containing carbon hydrogen.

After incubating for about 72 hours at 24 to 28 ° C with stirring, part of the culture becomes put into each of a large number of fermenting vessels, which are stirred, and an aqueous nutrient medium, the at least one normal peraffin having about 12 to 18 carbon atoms as the main source for contains assimilable carbon. The fermentation tanks operating at a speed of 650 up to 1750 rpm. be stirred and through which 2 to 8 liters of sterile air are passed per minute incubated for about 8 to 9 days at 24 to 25 ° C.

The amount of vaccine used in the different growth stages depends to a certain extent Degree on the density of cell growth. Generally about 5% is vaccine with good cell growth satisfactory for every stage of production.

The concentrations of D-mannitol, D-arabitol and i-erythritol are determined by a combination of determined colorimetric and thin-layer chromatographic determination methods. The total salary on polyol using a modification of the method described in J. Lab. CHn. Med. Vol. 54, p. 158 (1959) colorimetric method according to Bailey detected. The concentrations of i-erythritol and D-arabitol are determined following thin-layer chromatography with cellulose F as adsorbent and acetone-water (85:15) as solvent for development and after visualization with p-anisidine and subsequent spraying with periodate solution visually appreciated.

The D-mannitol is obtained from the entire fermentation broth by passing the broth through a centrifugal disk separator and first removing the yeast cells and then the remaining hydrocarbon from the aqueous phase. The cloudy aqueous phase is clarified by filtration and then deionized by passing it successively through a weak anion exchanger, a strong cation exchange resin and then a weak anion exchange resin. The clarified deionized broth is then concentrated to a solution under reduced pressure. Then allowed to crystallize with cooling. The crystalline D-mannitol is filtered off or centrifuged off. Additional crystal yields of D-mannitol are obtained by further concentration of the mother liquors.
The following examples explain the inventive method.

The strain Candida lipolytica ATCC No. 20297 is obtained by cultivating cells in a Mixture of skimmed milk and beef blood serum lyophilized.

Work cultures are prepared by dai3 one (25 mm x 150 containing 17 ml agar) periodically slant culture medium comprising using a sewing 17 ml agar) using a medium consisting of 0.5 ° / ₀ glucose, 0.3 ° / ₀ beef extract,

»Ο 0.5% yeast extract. 3.0 ⁰ ₀ Z peptone and 2 ° / ₀ agar subcultures generated.

Manufacture of the standard vaccine

One of these inclined culture media from Candida iipoly- »5 tica ATCC No. 20297 is washed with 20 ml of sterile distilled water. 0.2 ml of the cell suspension obtained is used to inoculate 10 ml of medium "A" (1% glycerol, 0.3% ring meat extract, 0.3% yeast extract and 0.5% peptone). Tubes (25 × 150 mm) containing 10 ml of medium and the vaccine are incubated for ^{20 hours at 28 ° C. with shaking.} The cultures obtained represent the standard vaccine.

Production of the basic medium "B"

This medium is formulated in such a way that it contains 2.0 g urea, 1.0 g (NH ₄ ) ₂ SO ₄ , 1.0 g CaSO ₄ · 2H ₂ O, 0.5 g (NH ₄ ) ₂ SO ₄ , 0.5 g K ₂ SO ₄ , 0.25 g MgSO ₄ • 7H ₂ O. 2.5 mg FeSO ₄ • 7 H _2, 0.10 mg MnSO ₄ • H ₂ 0.500 micrograms thiamine hydrochloride and 1.0 ml of a solution contains trace minerals. The main amount of the medium is adjusted to pH 3.5 and sterilized in the autoclave (the urea is sterilized and added separately). The trace element solution (1.0 ml) provides 1.0 mg CaCl ₂ -2H ₂ O and 100 micrograms each of CuSO ₄ · 5H ₂ O, CoCl ₂ · 6H ₂ O, ZnSO ₄ · 7H ₂ O, (NH ₄ ^ Mo ₇ O ₂₄ · 4H ₂ O and Na ₂ B ₄ O ₇ · 10H ₂ O.

example 1

The cells in the standard vaccine are washed twice with water and resuspended in water so that the final cell concentration is approximately 10 mg / ml. Flasks containing 20 ml of medium "B" and 4 ml of n-hexadecane are inoculated with 1.0 ml of the above cell suspension. The flasks are then incubated for 9 days at 24 to 25 ^° C. on a shaking device. The aqueous portion of the broth is separated from the cells and the remaining hydrocarbon by centrifugation. Based on the colorimetric determination and thin layer chromatography, the aqueous solution contains 34 mg / ml D-mannitol, <1 mg / ml D-arabitol and 3 mg / ml i-erythritol.

Example 2
1st stage of growth

300 ml Erlenmeyer flask, each containing 8 ml medium "A", 12 ml medium "B" and 4 ml n-hexadecane

5 6

are then allowed to crystallize with 1.0 ml of the above standard with cooling. The vaccine inoculated and on a shaker first yield of crystalline D-mannitol is 41 g. Incubated for 48 hours at 24 ° C. After recrystallization from water, these have ",". , Crystals a melting point of 168 ^c C (corrected), 2nd growth stage _{5 of the ham} mixing with your authentic sample Fernbach flask, the 20 ml medium "A", 380 ml of D-mannitol is not reduced. Further from medium "B" and 80 ml of mixed paraffins (8% of crystalline D-mannitol is obtained from HC ₁₁ H ₃₀ , 72% nC ₁₅ h ₃₂ , 15% nC ^ H ^), concentration of the mother liquors,
are each with 20 ml of the first growth stage. .
inoculated and 72 hours at 24 ° C on a Schottel ίο B e ι s ρ ι e IJ
device incubated. The procedure of Example 2 is repeated,,,. However, the mixture of n-paraffins is in place _in rroauktionsstute _{lemen nutrient medium} gi ^ e _calibrated amount by weight

4-1 fermentation vessel, which with a stirrer a mixture of equal parts n-dodecane,

equipped and 2000 ml of medium * B «and 15 n-tridecane, n-pentadecane, n-hexadecane and n-octa-

400 ml of mixed paraffins (as above) contain decan used, giving comparable results

are obtained with 100 ml of the second growth.

step vaccinated and 2i2 hours at 24 to 25 ° C. Example 4
broods. The fermentation tanks are equipped with a

Speed of 1750 rpm. stirred, and je ao The manufacture of the standard vaccine, the first

21 minutes of sterile air are passed through them. Growth stage and the second growth stage

_r . , are carried out as described in Example 2,

however, each mare at an incubation temperature of jvon

The entire fermentation broth is used to remove the 28 ° C. Each 480-Brunswick fermenters used, each of which leads ml of the second growth stage yeast cells and the remaining hydrocarbon * s are used to inoculate a plurality of 14-1-Newdurch a Zentrifugalscheibentrennvorrichtung overall. The cloudy aqueous phase is clarified by filtering 7 liters of medium "B" and 1400 ml of mixed paraffins and then deionized by adding them (as described in Example 2). The fermentation through a weak Ariionenauschharz, container is kept by cooling from the outside to 25 ⁰ C a strong cation exchange resin and a black and at a speed of 650 rpm. Ches anion exchange resin leads. 161 of the clarified stirred. At the same time, 8 liters of deionized broth per minute, the 12.5 mg / ml of D-mannitol, are de-aired through them. After 212 hours of incubation, the broth contains 12 mg / ml of D-mannitol, which is concentrated after dissolving 25% (weight / volume), which is obtained in Example 2, under reduced pressure.

Claims (2)

agreed widely available hydrocarbons claims: can be produced if Candida lipolytica ATCC No. 20297 is grown in an aqueous nutrient medium, 1. A process for the fermentative production of the at least one normal paraffin with about 12 to D-mannitol employing a strain of Candida, 5-18 carbon atoms as the main source of absorption thereby characterized in that one contains baren carbon. In this procedure, Candida lipolytica ATCC No. 20297 in one of the main product D-mannitol in aqueous nutrient medium that has at least a normal concentration such that it can be extracted directly males paraffin with about 12 to 18 carbon - a concentrate of the clarified and deionized atoms can be crystallized as the main source of assimilable carbon dioxide fermentation broth. contains lenstoff, cultivates and the formed The method according to the invention thus opens up D-mannitol directly from the clarified and deionic - a second route to fermentation production fermented fermentation broth wins. of D-mannitol compared to the well-known fermentation 2. The method according to claim 1, characterized by the characteristic production of D-Mannii from carbohydrate, that the materials used to make the D-Man. The main source of fuel in it nits used standard vaccine by any hydrocarbons used are in large 20 hour cultivation of Candida lipolytica ATCC volumes available inexpensively. No. 20297 in an aqueous nutrient medium when carrying out the invention 28 C. Procedure is Candida lipolytica ATCC No. 20297 20 cultured aerobically in a nutrient medium containing at least one normal paraffin for about 8 to 9 days 12 to 18 carbon atoms as the main source for contains assimilable carbon in intimate mixture with an aqueous phase, the assimilable The invention relates to fermentative nitrogen, minerals and other common nutritional processes for the production of D-mannitol under substances. In the method according to the invention Use of a Candida strain. amounts of D-arabitol and i-ery- The process according to the invention is characterized by the fact that the amount of D-mannitol formed is so low that with this process Candida is so great that the D-mannitol is produced by direct crystalline lipolytics ATCC No. 20297 in an aqueous nutrient sation pure form from the clarified and deionized medium, which at least one normal paraffin can be obtained with sated fermentation broth,
about 12 to 18 carbon atoms as the main source of In the nutrient medium, individual n-alkanes of Contains assimilable carbon, and breeds the dodecane to octadecane as the only hydrocarbons D-mannitol formed directly from the clarified and present. But also mixtures of coal deionized Fermentation broth wins. 35 hydrogens are usable, including crude D-mannitol is a non-hygroscopic white and semi-refined "material, however, at least some of these materials should have a pleasantly n-parafsweet taste, the sweetness of which is about 65% of the fin with a chain length of about 12 to 18 carbons. Sucrose is made up of atoms in food, the hydrocarbon content used to give consistency and in 40 chewable tablets such as vitamins or aspirin must be at least about 5 percent by weight for substantial amounts D-mannitol tablets useful as an extender. D-mannitol can be formed. If desired, amounts of finer amounts can be used to make synthetic resins and plasticizers up to 20 percent by weight. To make as well as an intermediate in the production of optimal results is preferred development of the well-known vasodilator D-Man- 45 content, however, about 15 percent by weight,
nitro hexanitrate can be used. Together with boron, the nutrient medium also contains the conventional acid, it is also used in the production of electrolytic borrowed sources for assimilable nitrogen, mineral dry condensers for use in radio emissions and other growth factors that are used in technology. aqueous phase. Urea is used as the source Most of the commercially available D-Man-50 are preferred for organic nitrogen, although also nits is made from sucrose. The sucrose becomes other materials, such as corn steep liquor, soybeans first used to make D-glucose and D-fructose hydrolyzate, amino acids and peptones sized and then reduced, after which you can become a mixture. It is known that certain from sorbitol and D-mannitol in a ratio of about 3: 1 vitamins and mineral cations and anions receives. The current relatively high 55 are favorable for the growth of yeast. One cares The cost of D-mannitol is based in part on the difficulty in finding a source of trace elements and vita, D-Mannitol in high purity from the mine, which has the right amount of this important sub-at the same time to separate formed sorbitol. stamping supplies. It is also known that strains of the genus Although any form of aerobic fermentation Candida from mixtures of carbohydrate substrates is satisfactory, controlled ventilation is used various polyhydric alcohols, such as glycerine, preferably, e.g. B. by stirring or by Hindurchi erythritol, Form D-arabitol and D-mannitol. In the duct of air. Since the hydrocarbon with the Literature is also described that Candida aqueous phase is not miscible, it is advisable Strains multiply in media that are the main source of it during fermentation in finely dispersed for assimilable carbon to hold hydrocarbons 65 shape, so that a large surface area of the contain. Hydrocarbon with the aqueous phase in It has now been found that D-mannitol comes into contact. This way it becomes an optimal low cost on an industrial scale from contact between the yeast cells, the aqueous phase