DE1442166B - Process for the production of fructose by microbiological conversion of sorbitol - Google Patents

Description

The invention relates to a process for the production of fructose by microbiological conversion of sorbitol, which is the conversion with certain fructose-forming microorganisms under cultivation the same carried out in the reaction medium.

The previously common processes for the production of fructose were carried out in such a way that acetic acid bacteria, such as Acetobacter suboxydans, in a medium composed mainly of mannitol were grown with the formation and accumulation of fructose in the medium (G. Bartrand: "Anal, de Chimie et Physique", 3 [VIII], p. 181 [1904]; Takahashi and As a i: "Nogei Kagaku Kaisha" [Journal of the Agricultural Chemical Society of Japan], 9, p. 396 [1935]); that cells of a certain belonging to the species Pseudomonas or Aerobacter Microorganism or an enzyme preparation of the cells of the microorganism to form a starting material from nannose or glucose were added to the isomerization (stereoisomerization) of the Bringing material with the formation of fructose (N. G. Va 1 e r ο η i et al .: »Journal of Biological Chemistry ", 218, p. 535 [1956]; O. M. Marshal et al .: "Sciense" 125, p. 648 [1957]; Tsumura and Sato: "Agricultural and Biological Chemistry", 25, p. 616 [1961]); or that to a starting material made from sorbitol, an enzyme preparation extracted from a specific acetic acid bacterium and an auxiliary substance accelerating the enzyme reaction was added, thereby producing fructose forms (J. T. Cummins et al .: »Journal of Biological Chemistry "224, p. 323 [1957]).

Of these methods, the one carried out using mannitol is extraordinary expensive because the cost of the raw material is quite high; that using cultured cells a specific microorganism or an enzyme preparation extracted from it requires 2 or 3 production steps and, moreover, has the disadvantage that the yield in fructose is generally relatively low.

Among the above-mentioned known methods is that using glucose or Mannose is carried out as the starting material, has the disadvantage that the inversion reaction does not run off completely and the fructose formed is difficult to separate from the remaining glucose. However, when a sugar alcohol such as mannitol or sorbitol is used as the starting material, the Easily separate the resulting fructose. Therefore, in this context, the use of the appropriate Alcohol is more beneficial than using a sugar.

Fructose is produced according to the usual processes using sorbitol as a starting material, by using an enzyme extracted from the cells of a particular microorganism, a diphosphopyridine nucleotide (DPN) and a substance such as B. pyruvic acid, the starting material sorbitol is added and mixed with this. In contrast, the method according to the invention becomes such carried out that one consisting of sorbitol as a carbon source and other suitable substrate materials Medium is inoculated directly with a specific microorganism and grown in it, whereby fructose is formed in the cultivation medium, which is then separated off and recovered. The inventive Process therefore has the decisive technical advantage that fructose is in only one stage is generated that the recovery of the fructose from the cultivation medium is proportionate goes easily and that the yield of fructose is higher than in previously known processes.

The present invention is explained in more detail below: As a fermentation process Sorbose fermentations have heretofore been made using sorbitol as a carbon source performed using Acetobacter or Gluconobacter. As is known, this is sorbose a starting material for the synthesis of vitamin C.

According to the invention, however, various samples of microorganisms have been isolated and discovered that these in a medium suitable for a fermentation process, in which sorbitol is used as the carbon source is used to form and accumulate fructose, various strains of the following have been found to be Species have this ability to oxidize. By carefully examining their microbiological Properties of these strains were called Bacillus megaterium, Pseudomonas fluorescens and Pseudomonas borepolis recognized.

1. Bacillus megaterium:
No. 3-8 C

No. 4-3 C

No. 6-IA-1

No. 7-IA-3

No. 11-3 C-3

No. 18-5A-2

No. 37-3 B (ATCC 15450)

2. Pseudomonas fluorescens:
No. 52-1C (ATCC 15453)

3. Pseudomonas borepolis:

No. 299-1 B-3 (ATCC 15452)
No. 242-9 B-5
No. 238-2 B-2

Since it was discovered that the abovementioned ability to form fructose

3 4

monas belonging to tribes, although the microaerobic conditions acidification. Optimal extent of this ability more or less strong cultivation temperature 27 to 37 ° C, at 40 ° C is not pronounced, the conclusion was drawn that this growth. Optimal pH range 6.5 to 7.2, wax oxidation capacity to a certain extent a range 5.5 to 7.6.
general property of these stems of the species is. 5 The bicrobiological properties of this micro-

As a result of further investigations this fer- organism were with those of similar micromentation became a strongly fructose-forming micro-organism, which is mentioned in Bergey's Manual of Deterorganism discovered, compared to the above-mentioned Bacillus minative Bacteriology, megaterium is somewhat similar, which, however, is found in one where the microcells found The microbiological properties of this organism are obviously different from all those previously known. That means the micro separates. The microorganism was therefore considered new Organism resembles the Bacillus megaterium in type and recognized as Bacillus fructosus nov. sp. microscopic observation and referred to in relation to (ATCC 15451).

physiological properties, differs depending on the- The invention therefore relates to the production of

but from this in the breeding and forms in a 15 fructose by microbiological conversion of

ordinary medium no spores. If a sorbitol, which is characterized in that one

Cultivation liquid of the microorganism to sterile- the conversion with a fructose-forming micro-

Sized quartz sand (performs twice under pressure at 120 ⁰ C organism, with a strain of the

3 hours sterilized) is given, under vermin genus Bacillus, Bacillus megaterium ATCC 15450,

derten pressure is dried and sealed to 20 Bacillus fruktosus nov. spec. ATCC 15451 or the

to obtain the strain, and 1 month at -4O ⁰ C genus Pseudomonas, Pseudomonas fluorescens

is left to stand, the microorganism forms on ATCC 15453 or Pseudomonas borepolis ATCC

■ '<Agar broth spore-forming colonies. Grow on agar-15452 in the reaction medium and add the fructose

If the microorganism is broken down, the microorganism dies within separated and wins.

a short time at room temperature, but is not 25 According to the method of the present invention

Change in how fructose is reduced will be used for fermentation

capability as in the case of Bacillus megaterium to medium by combining 5 to 10 ° / _o sodium

observe. aqueous sorbitol solution, 0.05 to 0.5 ° / ₀ of an organic

The microbiological properties of this micro or inorganic nitrogen source, such as yeast extract,

Organism are as follows: 30 meat extract, casein hydrolyzate, corn steep liquor,

....,. , τ,,, nitrate, ammonium salt or urea, and suitable

Microscopic observation · _{amounts of inorganic} salts, such as 0.05 to 0.2 ° / ₀

Rods, 1.0 to 1.5-4.0 to 5.0 μ with round potassium phosphate, 0.05 to 0.2 ° / ₀ magnesium sulfate and

Ends, singly in pairs or in short chains, of a small amount of another heavy metal salt

occurring. Granules observable in microbial cells. 35 produced and the medium obtained in this way

Grief negative. Not acid resistant. Spore formation adjusted to pH 6 to 8. After sterilizing the

depending on conditions, however, usually medium and incorporation form 1 ° / ₀ separately

they are difficult, 0.9 to 1.2 · 1.0 to 2.0 μ, are around sterilized calcium carbonate with the micro-

the center around or at the ends, the spore wall thin. Organism inoculated and the microorganism im

Sporongia not swollen. 40 medium grown.

_ ..,, A suitable temperature for breeding is

Breeding observation _{25 to} 350 _Q ^ _{breeding time {for one under} .

Gelatin rod culture: slow liquefaction. submerged culture is 2 to 4 days, whereas at

ί · Peptone agar nutrient broth, colonies: large circular areas, surface cultures 5 to 10 days are required.

completely coherent, smooth, soft, raised, 45 The amount of formed in the growth medium

non-spreading, dense, yellowish-brown. Fructose is made according to the sugar determination method

Peptone agar broth slopes: good growth, fa- determined by Bertrand, or according to the modi-

den-shaped, smooth, soft, opaque, glittering, non-fected method of S 0 m ο g y i.

coherent, yellowish brown. The fermentation yield, i.e. H. the definite

Glucose-agar-nutrient broth slopes: growth of sufficient 5 ° amount of fructose, reaches 80 to 85% based on the

fender, soft, glittering, cream-colored to yellow. Amount of soibit used.

Peptone nutrient broth: Slightly cloudy, not film-forming, the fructose formed is from the breeding

failed. medium obtained in the following way: The breeding potato: Good growth, soft, viscous, glittery. medium is filtered to remove residual calcium carbonate Potato agar slopes: good growth, soft, viscous, 55 and cells to remove the filtrate after setting

golden yellow. the pH to neutral under reduced

. Pressure to one fifth of the original volume

Physiological properties narrowed. For the concentrated filtrate is necessary

Nitrates are not reduced. Made from arabinose, if alcohol is added to make up the remaining soibit.

Xylose, glucose, fructose, galactose, lactose, cane precipitate, which is filtered off, whereupon the alcohol passes through

sugar, maltose, raffinose, inulin, starch, dextrin, distillation is removed. To the concentrated one

Glycerin, mannitol and sorbitol (with ammonium salts Filtiat is at low temperature for the purpose of formation

as a nitrogen source) acid is formed, but no gas. of calcium fructate added to milk of lime, which then

Acetylmethylcarbinol is not formed. Methyl red is obtained by filtration. The calcium fructate

reaction positive. Some hydrogen sulfide formation. 65 is suspended in water and carbon dioxide

No indole formation. Litmus milk is peptonized. directs to release fructose; the formed calcium

Catalysis positive. It is filtered off from sorbitol under aerobic conditions. The aqueous fructose solution

Large amounts of fructose are formed. Under is made by treatment with activated charcoal or with a

Purified ion exchange resin and then concentrated under reduced pressure to give fructose crystals to obtain.

The crystals were positive for the Selivanov reaction; it was chromatographed on paper using anisidine hydrochloride, forming a yellow spot that was suitable for Ketohexoses is characteristic (in the case of an aldose, the color is brown), and the Rf value agreed with that of fructose match. If the crystals were heated with phenylhydrazine in a boiling water bath, An osazon formed within only 6 minutes (Sobose does not form an osazon, not even at 15 minutes heating). The Osazon showed a melting point of 204 to 205 ° C and is thus with Fructoseosazon identical. If the osazone is mixed with that from fructose, the mixture does not show any Melting point depression. In addition, the aqueous solution of the crystals with yeast becomes rapid fermented (sorbose is not fermented), and when the crystallizate is mixed with lime it forms a complex salt. On the basis of these facts it was concluded that the crystallizate must be fructose.

The above discussion is directed to parts related to a process for making fructose in which the desired product is separated from the fermentation liquor and recovered. The invention also relates to a method for producing a fructose-containing sweetener, which consists in inoculating a medium with the microorganism and cultivating it in the medium, interrupting cultivation after 24 to 72 hours, ie if the fermentation yield: amount of fructose formed / sorbitol, for example Has reached 20 to 50 ° / ₀ , the intermediate culture liquid is filtered and the filtrate is purified by treatment with activated carbon or an ion exchange resin and then concentrated. In this case, the medium should only consist of inorganic salts, with the exception of sorbitol.

Since the sweet liquid obtained by the method described above still large amounts of Contains sorbitol, it is excellent as a moist sweetener. Sweeteners of this type were usually made by deliberately adding glucose or sucrose to the sorbitol incorporated. However, if fructose is incorporated according to the method according to the invention, that is obtained mixture very sweet, so that it forms an excellent wet sweetener that one has excellent taste.

The composition and conditions of the culture medium for the Fermentation studied in detail, and it was found that in a sorbitol medium that determined Contains amino acids as a source of nitrogen and certain heavy metals that accelerate fermentation will and fructose in a short time with high yield

ίο is formed.

The method according to the invention and the advanced results of the studies on which based on the present invention are explained in detail below:

In fructose fermentation, a medium containing inorganic nitrogen compounds as a nitrogen source generally gives low yields, whereas a medium containing corn steep liquor in addition to organic nitrogen sources gives much higher fermentation yield and speed. This effect is believed to be due to certain inorganic salts contained in the corn steep liquor. Based on this assumption, fermentation experiments were carried out using sorbitol media which, in addition to various nitrogen sources, contained a hydrochloric acid solution of the ash from corn steep liquor, which was obtained by evaporating corn steep liquor to dryness, roasting the residue at 600 ° C. to form an ash residue and dissolving the ash residue in one small amount of hydrochloric acid was obtained. The basic composition of the sorbitol media used in these experiments was 5% sorbitol, 0.1% KH ₂ PO ₄ and 0.05% MgSO ₄ -7 H ₂ O; the media was adjusted to pH 7.2. The amounts of the various nitrogen-generating substances to be added were determined beforehand so that the same cell propagation speed was obtained. The amount of corn steep liquor residue added was equal to 0.3% corn steep liquor. The cultivation was carried out in a shaking culture, Bacillus fructuosus nov. sp. (ATCC 15451) used. The amount of fructose formed was determined using the modified Somogyi method. The results obtained are summarized in Table 1.

Table 1 Fructose yield (based on sorbitol)

Nitrogen source

Breeding time 72 hours ! 4 hours 48 hours 7o % 0 /
/ 0 12.8 7.7 10.3 31.6 11.5 24.4 15.0 12.6 13.4 74.8 32.5 75.6 22.0 14.0 18.0 65.3 40.8 80.1 16.0 13.0 14.0 58.1 26.8 51.3 14.8 12.6 13.4 30.6 12.1 23.8 72.0 20.0 51.0

0.1% ammonium acetate.

+ Ashes

0.2% yeast extract

+ Ashes

0.3% casate

+ Ashes

0.3% meat extract

- ashes

0.15% peptone

+ Ashes

0.3% corn steep liquor

From the results of the above experiment it can be seen that the addition of ash residue alone to accelerate fermentation with regard to z. B. to the use of inorganic Nitrogen sources achievable values are not sufficient, although ash residue from corn steep liquor is evident shows an excellent fermentation-accelerating influence. When Casate (an enzymatic Casein hydrolyzate, manufactured by Toyo Vitamin Co. Ltd.) in association with ash was used, the yield is higher than if corn steep liquor was used alone. It It was therefore concluded that in fructose fermentation amino acids also have a fermentation-accelerating effect Effect result, in addition to the certain inorganic salts.

Then the above-mentioned hydrochloric acid solution of the ash residue was made from corn steep liquor treated with an ion exchange resin to break the solution into a cation and an anion fraction to subdivide. Both fractions were subjected to the fermentation test, and it was found that that only the cation fraction exerts this fermentation-accelerating influence, the anion fraction but not at all. Based on these findings, fermentations were carried out, wherein Media were used that contained Casate as a nitrogen source, which resulted in favorable results the tests in Table 1, and those low Quantities of various heavy metal salts have been added. As a result, it was found that. Media, which contain a salt of a heavy metal such as manganese, cobalt, copper or zinc, practically the show the same results as when dissolved ash residue is added to the medium. The relationship between the added amounts of heavy metal salts and the fructose yield is shown in Table 2 shown for manganese. The values listed in Table 2 were each obtained through 48 hours of cultivation receive.

Table 2

Manganese content Fructose yield, based on sorbitol in the medium Bacillus fructosus Bacillus megaterium ppm (ATCC 15 451) ° / o (ATCC 15 450) ° / " O 20.3 10.5 0.3 72.1 64.0 0.5 80.5 71.6 1.0 81.1 72.2 10.0 81.1 78.3 30.0 82.0 72.5 50.0 81.8 65.6 100.0 80.5 56.6 150.0 52.2 23.5 200.0 27.8 9.1

From the results shown in Table 2 it can be seen that the optimum in fructose fermentation The concentration range for manganese is about 0.3 to 100 ppm. However, other metals showed one relatively narrow range for optimal concentration, i.e. H. 0.5 to 1 ppm.

Since the fermentation-accelerating effect of metals was thus established, fermentation tests were carried out to investigate the synergistic effect of amino acids and metal salts on fermentation acceleration, using media which contained various nitrogen-supplying substances and to which MnSO ₄ · 4 H ₂ O was added so that the Mn concentration was 1 ppm. The experiments were carried out in practically the same manner as in the case of Table 1. The test results are summarized in Table 3.

Table 3 Fructose yield (based on sorbitol)

Nitrogen source 24 hours

Breeding period
48 hours

72 hours

0.1%

0.2% 0.4% 0.2%

0.1%

0.12%
0.4%

0.1%

0.02%
0.08%
0.05%
0.06%

0.3%

Ammonium acetate ...

+ Mn

Casate

+ Mn

Glutamic acid

+ Mn

Aspartic acid

+ Mn

Aspartic acid

Glutamic acid

+ Mn

Arginine hydrochloride.

Proline

+ Mn

Arginine hydrochloride

Proline

Aspartic acid ....

Glutamic acid

Glutamic acid + Mn

Corn steep liquor.

3
7th
4th

43
2

25th
6th

24

4th 30th

4th 32

45

25th

12th
30th
15th
81
14th
51
10
48

16
50

19th
56

80
51

17 56 28

20 80 19 79

23 77

26 80

23

76

209 546/532

As a result of these experiments it was found that effective amino acids arginine, proline, aspartic acid, Are glutamic acid, alanine, leucine and valine. Of these, L-glutamic acid, L-aspartic acid, L-arginine and L-proline as particularly accelerating fermentation recognized. If these four amino acids are used individually, this is the one achieved Fermentation speed slightly slower than in the case of the Casate, but if two or more of these amino acids are used at the same time, the fermentation rate is practically that same in the case of the Casate.

As stated above, the present invention aims at a process for the production of fructose, by adding one or more amino acids, L-glutamic acid, L-aspartic acid, L-arginine or L-proline, and substances containing these amino acids, such as Decomposition products of proteins, plant extracts, and microbial cell extracts and continue through Addition of a small amount of one or more inorganic substances, in particular manganese, Cobalt, copper and zinc salts, made into a sorbitol medium, with a microorganism of the Bacillus species, the Bacillus megaterium or Bacillus fructosus nov. sp. can be, inoculated and cultivates this microorganism, whereby fructose is formed and accumulated in the medium, and by recovering the fructose from the medium.

The amount of amino acid added to the medium used in the present invention is within the same range of amounts of nitrogen sources that is added to a conventional fermentation medium. Is the amount of amino acids in excess, the fermentation yield is reduced due to excessive cell reproduction. In addition, the amount of amino acids depends on the concentration of sorbitol, the source of carbon. The suitable amount of amino acid is about 0.6% when the medium has a sorbitol concentration of 15% and contains casate as a nitrogen source; it is around 2 to 3% in the case of one Medium with a sorbitol concentration of less than 15%

example 1

being a slurry of calcium fructa formed. The calcium fructate was filtered off with suction and the filter cake obtained was cooled to below 5 ° C Water washed. The calcium fructate was then placed in 200 ecm cold water suspended and initiated after adjusting the pH to about 7.0 carbon dioxide, calcium carbonate itself taking leave. The precipitate was removed by filtration to give a free fructose solution

ίο was.

The solution was adjusted to pH 4.5 with hydrochloric acid and decolorized by adding activated charcoal and then treated with an ion exchanger to remove inorganic substances. The received colorless transparent liquid had a fructose content of 5.2% and a volume of about 400 ecm. The liquid was concentrated under reduced pressure; it became about 10 g of crystalline fructose receive.

20th

Example 2

A medium having the same composition and volume as in Example 1, was inoculated with a strain of Pseudomonas borepolis No. 299-IB-3 (ATCC 15452) and the microorganism grown at 30 ° C for 3 days, with a maximum of 36% fructose, based on sorbitol, was formed.

After the cultivation was completed, about 1.5 liters of the cultivated medium was collected, filtered, neutralized and concentrated under reduced pressure to a volume of about 100 ecm. To the narrowed An excess of alcohol was added to the broth to precipitate residual sorbitol which had passed through Filtration was removed. The filtrate was distilled to remove the alcohol. The remaining one Liquid was cooled to below 5 ° C and in the same manner as in Example 1 to give Calcium fructate, fructose release, treatment with activated carbon and ion exchange resin purified, whereby 400 ecm of a 5.5% fructose solution were obtained. This solution turned out to be about 11g crystalline fructose obtained.

45

50 ecm of a medium with a pH of 7.2 and composed of 5% sorbitol, 0.1% casein hydrolyzate, 0.1% ammonium chloride, 0.1% potassium phosphate and 0.05% magnesium sulfate was placed in 500 cc shake flasks and sterilized. To each Medium was added to 1% separately sterilized calcium carbonate. The medium was with a Bacillus megaterium strain No. 37-3 B (ATCC 15450) inoculated and grown at 30 ° C with shaking. Reached after 3 days of cultivation the fructose formation a maximum of 51% based on sorbitol.

After the cultivation was completed, the cultivation medium became filtered to remove the calcium carbonate and the cells and adjusted to pH 7.0. 1 liter of the medium was concentrated under reduced pressure to a volume of 200 ecm. the concentrated broth was cooled to below 5 ° C. To the cooled concentrate were under Stirring 50 ecm of a 20% calcium oxide milk, which was also cooled to below 5 ° C, added,

Example 3

50 ecm each of a medium with a pH of 7.0, which consists of 5% sorbitol, 0.4% corn steep liquor, 0.1% potassium phosphate and 0.05% magnesium sulfate were placed in 500 cc shake flasks and sterilized. Each medium was provided with 0.2% separately sterilized calcium carbonate. That on this Medium prepared in this way was mixed with Bacillus fructosus nov. sp. (ATCC 15451) and in Cultured at 30 ° C with shaking. After 3 days of cultivation, the amount formed reached Fructose 85% based on sorbitol.

The cultured medium was filtered and drained to remove calcium carbonate and cells pH 7.0 adjusted. 1 liter of the filtrate was collected and reduced under reduced pressure Volume restricted to 200 ecm. The concentrated broth was cooled to below 5 ° C. To the chilled 150 ecm of a 20% calcium oxide milk, which is also under 5 ° C was added, forming a slurry of calcium fructate. The

11 12

Calcium fructate was sucked off and the resin obtained), Amberlite IR 120 (strongly acidic cation filter cake with water, the polystyrene sulfonic acid-based exchanger cooled to below 5 ° C, from was washed. The Calcium Rohm & Haas Co., V. St. A.), Amberlite IRA-68 fructate suspended in 200 ecm cold water and (weakly basic anion exchange resin on Polynach Adjustment of pH to 7.0 carbon dioxide based on styrene-tertiary-amine, from Rohm & Haas Co., conducts, causing calcium carbonate to be precipitated V. St. A.) and a mixture of Amberlite IR 120 would. The precipitate was removed by filtration and Amberlite IRA 411 (strongly basic anions, so that a free fructose solution was obtained from polystyrene-based exchangers with quaternary amines. monium groups, from Rohm & Haas Col, V. St. A.)

The solution was treated to pH 4.5 with hydrochloric acid in order to adjust inorganic salts and organic acids, and with the addition of activated charcoal it was decolorized and protein and coloring substances were removed. In addition, for removing inorganic substances, 3.3 liters of the treated solution including was treated with an ion exchange resin. The conservation borrowed washing solution obtained, 18.2 ° / ₀ fructose enttene colorless transparent liquid had a thought. The liquid was reduced to a fructose content of 9.2 ° / ₀ and had a volume of 15 pressure further to a volume of about 1 liter of about 400 ecm. The liquid was concentrated under a viscous syrup. The syrupy liquid distilled under reduced pressure; About speed was obtained with methanol and 2 g of fructose inoculum 21 g of crystalline fructose. crystals intimately mixed and the mixture at one

Keep cool place for crystals to form

^Example4 let 20. The crystals were separated by filtration

and dried, with a yield of 420 g

The same strain as in Example 3 was obtained.

using the same manner as in Example 3 The crystals were positive to the reaction

of from 5 ° / ₀ sorbitol, 0.1 ° / ₀ ammonium acetate or by Selivanov and were papierchromatographiert

Ammonium chloride, 0.1% potassium phosphate, 0.05 ° / ₀ 25 using anisidine hydrochloride, whereby

Magnesium sulfate and 0.1 ° / ₀ CaCO ₃ is existing spots formed with a yellow color, which for keto

Cultured medium of pH 7.0 with shaking. hexoses are characteristic (in the case of an aldose

After 48 hours of cultivation, the cultivation became brown), the Rf value agreed with that for

moieties, where the fermentation yield 42 ° / ₀ fructose match. The crystals were made together

reached. The culture fluid was collected with phenylhydrazine on the boiling water bath

filtered, treated with activated charcoal and heated, formed within just 6 minutes

Subjected to ion exchange resin treatment and an osazon (sorbose forms even at 15 minutes

then to a colorless, transparent viscous heating no Osazon). The Osazon had one

Solution with a sorbitol content of about 30% melting point of 204 to 2O5 ° C and was with the

concentrated that has an excellent taste 35 osazon identical to the fructose. If the Osazon

had. with which the fructose was mixed shows this

resulting mixture no melting point depression.

Example 5 A f _u basis of these facts, it was found that the

Crystals consisted of fructose.

750 g sorbitol, 30 g casate, 5 g KH ₂ PO ₄ , 2.5 g 40

MgSO ₄ · 7 H ₂ O and 0.05 g of MnSO ₄ · 4 H ₂ O were in B eis ρ ie 1 6
distilled water dissolved in 5 liters of the solution

was then ^{sterilized at 120 °} C. for 10 minutes. The 750 g of sorbitol, 30 g of Casate, 5 g of KH ₂ PO ₄ , 2.5 g of the medium prepared in this way were immersed in a MgSO ₄ · 7 H ₂ O and 0.05 g of MnSO ₄ · 4 H ₂ O were 10 liters The fermenter with a capacity was dissolved together with 50 g of 45 in distilled water to make 5 liters of solution, the dried and sterilized calcium carbonate and then sterilized at 120 ^° C. for 10 minutes. 200 ecm of an inoculation solution, obtained by culturing the Bacillus for 6 hours while shaking the medium prepared in this way, Bacillus megaterium No. 37-3 B (ATCC 15450) fructosus nov. sp. (ATCC 15451) was obtained in a medium in a fermentor in the same manner as in that of 2 ° / ₀ sorbitol, 0.5% 50 Example 5 Maisquell- fermented. Was after 92 hours of culture liquid and 1 ° / ₀ CaCO _3. The cultivation time reached the determined content of fructose microorganism was 118 mg / ml under aerobic conditions. The fermented medium was ^{centrifuged to remove cells at 30 °} C. with stirring (430 rpm) and aeration, and carried out with hydrochloric acid (1 liter per minute). adjusted to pH 6.5 and the lower

After an hour of cultivation, the fructose flake began to be removed by filtration. The filtrate was

formation, and after 72 hours the concentration reached- concentrated and treated with ion exchange resins,

tiation 123 mg / cc. After that, the formed began being about 3 liters of a transparent liquid

Decrease amount of the culture was thus obtained with a fructose content of 19.3 ° / ₀

interrupted. The culture liquid was used in order to be. The liquid was reduced under

Removal of cells and calcium carbonate in a .60 pressure further concentrated to a volume of about

Clarifier treated and the obtained transparent 1 liter. The concentrated liquid was with

Liquid mixed under reduced pressure to one volume of methanol and 2 g of fructose seed crystals

of about 2 liters concentrated. The concentrate was put in a cool place to crystallize out the

then one after the other with Centranol 291 (decolorization fructose. Yield: 395 g.

Claims (3)

Patent claims: 1. Process for the production of fructose by microbiological conversion of sorbitol, thereby characterized in that the conversion is carried out directly with a fructose-forming Microorganism carries out, whereby a microorganism strain of the genus Bacillus, Bacillus megaterium ATCC 15450, Bacillus fruktosus nov. sp. ATCC 15451 or the genus Pseudomonas, Pseudomonas fluorescens ATCC 15453 or Pseudomonas borepolis ATCC 15452 grows in the reaction medium. 2. The method according to claim 1, characterized in that the sorbitol is used as a carbon source, a suitable nitrogen source, inorganic salts and optionally a fermentation accelerator containing medium inoculated with the fructose-forming microorganism and the microorganism breeds in this medium. 3. The method according to claim 1 and 2, characterized in that the microbiological Conversion is interrupted when the amount of fructose formed has a value of about 20 to 50%, based on sorbitol.