DE1442166C - 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 Chimieet Physique", 3 [VIII], p. 181 [1904]; 1 akahashi and A s 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. Valeroni 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 for a starting material made from sorbitol, an enzyme preparation consisting of a specific acetic acid bacterium, and one that accelerates the enzyme reaction Auxiliary substances were added, whereby fructose is formed (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. thats why

ίο 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 hereinafter described in

explained in more detail: As a fermentation process using sorbitol as a carbon source Sorbose fermentations were previously carried out, with Acetobacter or Gluconobacter were used. As is known, this sorbose is 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-IC (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, it was concluded 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 (twice under pressure at 120 ° C organism, using a strain of

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 maintain the strain, and 1 month at -40 ° 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 diet breaks 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 ° / ₀ sodium

observe. aqueous sorbitol solution, 0.05 to 0.5% of an organic one

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,

. Nitrate, ammonium salt or urea, and suitable

Microscopic observation. Amounts of inorganic salts, such as 0.05 to 0.2%

Chopsticks, 1.0 to 1.5-4.0 to 5.0 μ with round potassium phosphate, 0.05 to 0.2% 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 produced 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% 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.

,,, Is a suitable temperature for the cultivation

Breeding observation _{25 bJs} 350 _{c> d Depending on the} breeding period for a sub-

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

Peptone agar broth, colonies: large circular areas, surface cultures 5 to 10 days are needed.

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-fied process by Somogyi.

coherent, yellowish brown. The fermentation yield, d. H. the definite

Glucose-agar-nutrient broth slopes: growth of sufficient 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 Α garhangs: 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 filtrate is used 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, whereby a yellow spot was formed which is characteristic of ketohexoses (in the case of an aldose, the color is brown), and the Rf value agreed with that of fructose. 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, even if heated for 15 minutes). Osazon had a melting point of 204 to 205 ^° C. and is therefore identical to fructoseosazon. If the osazone is mixed with that from fructose, the mixture shows no depression of the melting point. In addition, the aqueous solution of the crystals with yeast is rapidly fermented (sorbose is not fermented), and when the crystals are mixed with lime, a complex salt is formed. On the basis of these facts it was concluded that the crystallizate must be fructose.

The above discussion focuses on parts that relate to a process for making fructose set up the desired product from the fermentation liquid separated and recovered. The invention also relates to a method for the preparation of a fructose-containing sweetener, which consists in a medium with the Inoculates microorganism and cultivates it in the medium, interrupting cultivation after 24 to 72 hours, i.e. when the fermentation yield: amount of fructose formed / sorbitol, has reached about 20 to 50%, the intermediate culture liquid is filtered and the filtrate is treated with activated charcoal or a Ion exchange resin cleans and then concentrated. In this case the medium should only consist of inorganic Salts exist, 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 yields inorganic nitrogen compounds as a nitrogen source contains, in general, low yields, whereas a medium containing corn steep liquor besides contains organic nitrogen sources, an essential

This results in higher fermentation yield and speed. It is assumed that this effect is due to certain inorganic salts which are contained in the corn steep liquor. Starting from this assumption, fermentation experiments were conducted using Sorbitmedien containing in addition to various sources of nitrogen a hydrochloric acid solution of ash from corn steep liquid obtained by evaporation of the corn steep liquor to dryness, roasting of the residue at 600 ⁰ C to form an ash residue and dissolving the ash residue in a 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 previously determined so that the same cell propagation rate 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, as a microorganism 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 / n "/ 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.S 51.3 14.8 12.6 13.4 30.6 12.1 23.8 72.0 20.0 51.0

0.1% ammonium acetate.

-r ashes

0.2% yeast extract

+ Ashes

0.3% casate

~ j- ashes

0.3% meat extract ....

; ash

0.15% peptone

; ash

0.3% iVlai.sqiicllliquid

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 The values that can be achieved by nitrogen swelling 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 when using "corn steep liquor alone. Es It was therefore concluded that in fructose fermentation amino acids also have a fermentation-accelerating effect Effect, in addition to the inorganic salts determined by t5.

Then the above-mentioned hydrochloric acid solution of the ash residue 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 added small amounts of various heavy metal salts became. As a result, it was found that media containing a heavy metal salt such as manganese Containing cobalt, copper or zinc, show practically the same results as if the medium dissolved ash residue is added. The relationship between the amounts of heavy metal salts added and the fructose yield is shown in Table 2 for manganese. Those listed in Table 2 Values were each obtained by culturing for 48 hours.

Table 2

Manganese content Fructose yield, based on sorbitol in the medium Bacillus fructosus Bacillus megaterium ppm (ATCC 15 451) ° / o (ATCC 15 450) »/ ο 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%

0.12%
0.4%

0.02%
0.08%
0.05%
0.06%

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 swelling liquor. ·.

3
7th
4th

43
2

25th
6th

24

4th
30th

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

309 025/112

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,! - 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 Casate as a nitrogen source contains; it is around 2 to 3% in the case of a medium with a sorbitol concentration below 15%.

example 1

50 ecm each of a medium that had a pH value of 7.2 and consisted of 5% sorbitol, 0.1% casein hydrolyzate, 0.1% ammonium chloride, 0.1% potassium phosphate and 0.05% magnesium sulfate were used in 500 -ccm shake flask filled and sterilized. 1% separately sterilized calcium carbonate was added to each medium. The medium was inoculated with a strain of the Bacillus megaterium no. 37-3 B (ATCC 15450) grown at 30 ⁰ C with shaking. After 3 days of cultivation, fructose formation reached a maximum of 51% based on sorbitol.

After the completion of the cultivation, the cultivation medium was filtered to remove calcium carbonate and 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 with stirring, 50 cc of a 20% Caiciumoxydmilch, which had also been cooled to below 5 ^U C, is added.

whereby a slurry of calcium fructa was formed. The calcium fructate was sucked off and de: The filter cake obtained is washed with water cooled to below 5 ° C. After that the Cakiumfruktat was suspended in 200 ecm cold water and after adjusting the pH to about 7.0 Carbon dioxide introduced, with calcium carbonate separated. The precipitate was collected by filtration removed to give a free fructose solution.

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.

20 B ice ρ ie! 2

A medium having the same composition and the same volume as in Example 1 was-IB-299 3 inoculated with a strain of Pseudomonas borepolis no. (ATCC 15452) and cultured, the microorganism at 30 ⁰ C for 3 days, with a maximum of 36% fructose, based on sorbitol, was formed.

After the completion of the cultivation, about 1.5 liters of the cultivated one was obtained. Medium 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 placed in the same way as in example 1 with formation of calcium fructate, release of fructose, treatment Purified with activated charcoal and ion exchange resin, creating 400 ecm of a 5.5% fructose solution were obtained. About 11 g of crystalline fructose were obtained from this solution.

45 B e i s ρ i e 1 3

50 ecm each of a medium of pH 7.0, which consisted of 5% sorbitol, 0.4% corn steep liquor, 0.1% potassium phosphate and 0.05% magnesium sulfate, were filled into 500 ccm shake flasks and sterilized. Each medium was provided with 0.2% separately sterilized calcium carbonate. The medium prepared in this way was mixed with Bacillus fructosus r.ov. sp. (ATCC 15451) inoculated and ^{grown at 30 0} C with shaking. After 3 days of cultivation, the amount of fructose formed reached 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 cooled one 150 ecm of a 20% calcium oxide milk, which is also under 5 ° C was added, forming a slurry of calcium fructate. The

Calcium fructate was filtered off with suction and the filter cake obtained was washed ^{with water which had cooled to below 5 ° C.} The calcium fructate was then suspended in 200 ecm of cold water and, after the pH had been adjusted to 7.0, carbon dioxide was passed in, whereby calcium carbonate was precipitated. The precipitate was removed by filtration to give a free fructose solution.

The solution was adjusted to pH 4.5 with hydrochloric acid and decolorized with the addition of activated charcoal also treated with an ion exchange resin to remove inorganic substances. The colorless transparent liquid obtained had a Fructose content of 9.2% and had a volume of about 400 ecm. The liquid was taking distilled under reduced pressure; about 21 g of crystalline fructose were obtained.

B e i s ρ i e 1 4

The same strain as in Example 3 in the same manner as in Example 3 using a 5% · sorbitol, 0.1 ° / ₀ ammonium acetate or ammonium chloride, 0.1 ° / o potassium phosphate, 0.05% magnesium sulfate, and 0 , 1% CaCO ₃ existing medium of pH 7.0 cultured with shaking. After 48 hours of cultivation, the cultivation was stopped, and the fermentation yield reached 42%. The culture liquid was collected, filtered, subjected to the treatment with activated carbon and the ion exchange resin treatment, and then concentrated into a colorless, transparent, viscous solution having a sorbitol content of about 30% and having an excellent taste.

B e i s ρ i e 1 5

75Og sorbitol, 30g Casate, 5 g KH ₂ PO _4, 2.5 g MgSO ₄ ■ 7H ₂ O and 0.05 g of MnSO ₄ · 4 H ₂ O were dissolved in distilled water to 5 liters of solution, which then at 120 ° C Was sterilized for 10 minutes. The medium prepared in this way was poured into a 10 liter fermenter together with 50 g of dried and sterilized calcium carbonate and 200 ecm of an inoculation solution, which was cultivated for 6 hours while shaking the Bacillus fructosus nov. sp. (ATCC 15451) in a medium consisting of 2% sorbitol, 0.5% corn steep liquor and 1% CaCO ₃ . The cultivation of the microorganism was aerobically at 3O ⁰ C with stirring (430 rpm) and aeration is performed (1 liter per minute).

After an hour of cultivation, the formation of fructose began, and after 72 hours the concentration reached 123 mg / cc. After that, the educated began Decrease in quantity, the breeding was therefore interrupted. The culture liquid was used for Removal of cells and calcium carbonate treated in a clarifier and the obtained transparent Liquid concentrated under reduced pressure to a volume of about 2 liters. The concentrate was then successively with Centranol 291 (decolorizing resin), Amberlite IR 120 (strongly acidic cation exchanger based on polystyrene sulfonic acid, from Rohm & Haas Co., V. St. A.), Amberlite TRA-68 (weakly basic anion exchange resin based on polystyrene tertiary amine, von Rohm & Haas Co., V. St. A.) and a mixture of Amberlite TR 120 and Amberlite IRA 411 (strongly basic anion exchanger based on polystyrene with quaternary ammonium groups, from Rohm & Haas Col, V. St. A.)

ίο treated to remove inorganic salts, organic acids, Remove protein and staining substances. There were 3.3 liters of the treated solution inclusive Wash solution obtained containing 18.2% fructose. The liquid was reduced under Pressure further concentrated to a volume of about 1 liter to a viscous syrup. The syrupy liquid was intimately mixed with methanol and 2 g of fructose seed crystals and the mixture on a kept in a cool place to allow crystals to form. The crystals were separated by filtration and dried, yielding 420 g.

The crystals were positive to the Selivanov reaction and were paper chromatographed using anisidine hydrochloride, forming spots of yellow color, which are for ketohexoses are characteristic (in the case of an aldose, the color is brown), the Rf value agreed with that for Fructose. The crystals were placed on a boiling water bath along with phenylhydrazine When heated, an osazone formed within just 6 minutes (sorbose also forms after 15 minutes Do not heat Osazon). The osazon had a melting point of 204-205 ° C and was with the Osazon identical to fructose. If the osazon was mixed with that of the fructose, that shows resulting mixture no melting point depression. On the basis of these facts, it was found that the Crystals consisted of fructose.

Example 6

750 g sorbitol, 30 g Casate, 5 g KH ₂ PO ₄ , 2.5 g MgSO ₄ · 7 H ₂ O and 0.05 g MnSO ₄ · 4 H ₂ O were dissolved in distilled water to make 5 liters of solution, which then · Have been sterilized at 120 ° C for 10 minutes. Using the medium thus prepared, Bacillus megaterium No. 37-3 B (ATCC 15450) was fermented in a fermenter in the same manner as in Example 5. After 92 hours of cultivation, the fructose content found reached 118 mg / ml. The fermented medium was centrifuged to remove cells, adjusted to pH 6.5 with hydrochloric acid and the precipitate which formed was removed by filtration. The filtrate was concentrated and treated with ion exchange resins to obtain about 3 liters of a transparent liquid having a fructose content of 19.3%. The liquid was further concentrated under reduced pressure to a volume of about 1 liter. The concentrated liquid was mixed with methanol and 2 g of fructose seed crystals and placed in a cool place for the fructose to crystallize out. 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 is around 20 to 50%, based on sorbitol.