FI64641C - FOERFARANDE FOER KONTINUERLIG FRAMSTAELLNING AV ISOMALTULOS FRON SACKAROS MED TILLHJAELP AV ISOMALTULOSBILDANDE MIKROOR GAISMER - Google Patents

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^ 11 UTLÄGGN, NGSSKRlFT 64 641 C (4S) ratonlti r.yjr.:..t'y 12 12 1933 '* Patent Eeddelat (51) Ky.ik. ^ nt.a.3 c η 2 P 19/12 / / C 07 H 3/04-FINLAND - FINLAND (21) P * tenttlh »lt« mu «- Pttentveökntnj 782? 68 (22) H * lceml * p» lvl - Antöknln (td «| 11.09.78 (23) Alkupllvi —Giltl | head «f 11.09.78 (41) Become Public - Blivlt offontllg 03.79

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Ä. . (44) Date of competition and issue. - ,, πο o,

Patent · och registerstyrefsen Antflkan utltgd och ucl. * Krift «n pubticend jx. υο. oj (32) (33) (31) requested front »» Begird prlorltet 13.09.77 lU.02.78 Federal Republic of Germany-Förbunds-republiken Tyskland (DE) P 27U1197-8, p 2806216.0 (71) Bayer Aktiengesellschaft, Leverkusen, Federal Republic of Germany- Förbundsrepubliken lyskland (DE) (72) Wulf Crueger, Erkrath, Lore Drath, Worms am Rhein, Mohammed Munir,

Obrighcim, Federal Republic of Germany-Dörbundsrepubliken Tyskland (DK) (7U) Qy Kolster Ab (5U) Method for the continuous production of isomaltulose from sucrose by microorganisms forming isomaltulose

The invention relates to a process for the continuous production of isomaltulose from sucrose by means of isomaltulose-forming microorganisms which are cultured in a sucrose-containing nutrient broth containing vegetable juices.

Isomaltulose is an intermediate in the preparation of glucopyranosido-1,6-mannitol (DE-OS 2 520 173) and glucopyranosido-1,6-sorbitol (isomaltitol, DE-PS 2 2X7 628). Both substances can be used as sugar substitutes.

It is known from German patent publication 1,049,800 that sucrose is enzymatically converted to isomaltulose. The required enzyme is derived from microorganisms. In addition to Protaminobacter rubrum, sucrose is converted to isomaltulose by other bacteria such as Ervinia carotovora, Serratia marcescens, Serratia plymuthica and 2,64641

Leuconostoc mesentheroides (b. Schmidt-Berg-Lorenz, W. Mauch, Zeit-schrift fur die ZuckerIndustrie, 14, 625-627 (1964); FH Stodola, 126th Meeting Amer. Chem. Soc., Sept. 1954> Abstracts of Papers , s.

5D; W. Mauch, S. Schmidt-Berg-Lorenz, Zeitschrift fur die Zuckerinustrie, 14, 309-315 and 375-383 (1964).

In addition, it is known from German patent publication 2,217,628 that the enzymatic conversion of sucrose to isomaltulose can be carried out batchwise or continuously with a 15-40% solution with vigorous stirring under aerobic conditions at a temperature of 20-37 ° C. At the end of the enzymatic reaction, according to said patent, the bacterial suspension is isolated by centrifugation and reused six times. According to the same patent, a continuous sucrose conversion reaction is carried out by culturing the bacteria on a special nutrient medium and continuously removing them. The enzymatic reaction takes place in another boiler cascade system with a bacterial culture and a thick juice or other sugar-containing solution obtained from sugar production.

But growing microorganisms separately from the enzymatic conversion reaction of sucrose is unsatisfactory for several reasons: a) Microorganisms are grown in their own medium, which increases the cost.

(b) Irrespective of the reactor boiler system, the culture medium and also the fermentor in which the enzymatic reaction takes place must be sterilized and kept sterile.

(c) The cultivation of micro-organisms in a nutrient solution other than the subsequent reaction solution runs the risk of selecting the micro-organisms for fermentation according to criteria other than the maximum reaction ratio.

(d) The isolation and re-use of micro-organisms in enzymatic further processing is associated with a high risk of infection.

It has now been found that the cultivation of isomaltulose-forming microorganisms and the simultaneous conversion of sucrose to isomaltulose can be carried out continuously.

By isomaltulose-forming microorganisms is meant herein primarily Protaminobacter rubrum and in addition Serratia olymuthica, Serratia marcescensia, Erwinia carotovora and Leuconostoc mesentheroides.

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It was surprising and unexpected that when isomaltulose-forming microorganisms, e.g. Protaminobacter rubrum (CBS 574 77), which convert sucrose to isomaltulose during their growth, in sucrose-containing vegetable juices, which are thus formed as an intermediate in e.g. cane and beet sugar production, are more expensive. than pure sucrose solution, particularly good yields are obtained at certain dilution rates. In this case, the growth of the organisms and the reaction rate are linked so that the overall process, i.e. the growth of microorganisms and the conversion of sucrose to isomaltulose, can be monitored by various critical parameters such as CCl 2 and

The method according to the present invention is characterized in that after a sufficient cell density has been reached, the culture is continued continuously, with sucrose-containing solutions being added continuously at dilution rates of 0.05-0.3 h "*, preferably 0.2 h and an equal amount of culture solution. is continuously removed, after the isomerization of the sucrose, the mass of microorganisms is separated from the solution and the solution is purified and / or crystallized in a known manner.

The method has the following additional advantage. When sucrose is converted to isomaltulose, small amounts of fructose and glucose are formed, which act as a carbohydrate source for the metabolism of microorganisms. At the same time, these unwanted by-products continue to be broken down.

For continuous isomaltulose fermentation, sugar mill thin juice / thick juice and / or thin juice / clarifier mixtures with a dry matter content of 5-30%, preferably 20-27%, are used as starting material. The sucrose content, calculated on the dry weight, is 90-98%, preferably 94-96%.

In order to achieve optimum growth of the bacterial culture, phosphate ions are added in the form of salts, e.g. (NH 2 O 2 O 2 or HP HPO 2) at a concentration of 0.05 to 1 g / l, preferably 0.1 to 0.5 g / l. Normally by-products of sugar production after juice purification still contain 40-150 mg / 100 g of residual calcium oxide in the dry matter.

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With the addition of PO 4 ions, this residual calcium oxide is precipitated as all 4,64641 sodium phosphate and thus part of the added phosphate is consumed for cell proliferation.

Thus, it is advantageous to remove the hardness from the 3-sugar solution to be fermented by a base ion exchanger, whereby the amount of PO ions to be added can be reduced.

The nutrient solution is sterilized in batches or continuously. There is no need to adjust the pH because the pH of the sugar solution used is 7.5-8.5.

The fermentor is inoculated with 0.1-10% inoculum cultured in the same nutrient solution in Erlenmeyer flasks on a shaker and incubated at 18-32 ° C, preferably 30 ° C, in an aeration ratio of 0.2-1.0, preferably 0.4 volumes of air / space a portion of nutrient solution for x minutes with adequate stirring.

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When a sufficient bacterial cell concentration has been reached, e.g. 10 bacteria / ml, the fermentation is carried out continuously, i. per unit time, remove the culture medium and add the same amount of new, sterile and uninoculated nutrient solution from the storage tank. At a dilution rate according to the invention of 0.05 to 0.3, preferably 0.2 per hour, the bacteria are not washed away. Bacteria grow so rapidly in the "steady state" that sucrose is converted to 90-100% isomaltulose. In general, stabilization of the pH during the reaction is not necessary.

The dilution rate and thus the removal of the culture medium from continuous fermentation is controlled e.g. with sterilizable oxygen electrodes by determining the oxygen partial pressure of the culture medium or the CCl concentration of the exhaust air or other physiological variables. An additional possibility to control continuous fermentation is to monitor the conversion of sucrose to isomaltulose by determining the reduction capacity of the fermentation broth. (It is known that isomaltulose has a reducing ability of 52% of the value of glucose, and sucrose has no reducing properties at all). The conversion of sucrose to isomaltulose can also be monitored by quantifying isomaltulose in addition to sucrose by high performance liquid chromatography.

The microorganisms are then isolated from the nutrient solution removed from the fermentor, e.g. by centrifugation, and further processed. The bacterial mass isolated by centrifugation is slurried a second time in water and centrifuged. It can be used e.g. cattle feed.

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If the culture solution removed from the fermentor has been allowed to react only e.g. 90%, the microorganisms carry out a complete reaction without adding more oxygen to the centrifugal conduction system.

The essential advantages of the present invention can be seen from the yields calculated on the basis of the amount of raw material required as well as the volume / time yields: column 5, lines 12 and 42 of DE 2 217 628 show that 1.8 kg of sucrose are obtained. isomaltulose. This corresponds to a yield of 45.0% calculated on the amount of sucrose fed.

By the process of the present invention, 2.6 kg of isomaltulose are obtained from 4 kg of sucrose. This corresponds to a yield of 65.0% based on the amount of sucrose fed, i.e. the yield is considerably higher than in the previous method.

According to the method of DE-A-2,217,628, the time required to prepare 1.8 kg of isomaltulose is 12 hours (column 5, lines 11-42). The amount of sugar solution to be fermented is 20 kg, which at a concentration of 20% by weight corresponds to a volume of 18.47 liters (density 1.083). This gives a volume / time yield of 8.1 g x 1 x x h -1.

According to the present invention, for example, 200 l of a sucrose solution with a dry matter content of 25% by weight and a purity of 96% are fermented. 40 liters of product per hour are continuously removed from the fermenter, and at the same time the same amount of fresh sucrose solution (dilution rate 0.2 h) is fed to the fermenter. This means that 40 liters of sucrose solution are treated per hour. The amount of sucrose fed at said concentration and purity gives 10.6 kg x h. This corresponds to 6.8 kg of crystallized isomaltulose with a purity of 99.9% per hour. Then the volume / time yield -1 -1 si becomes 34 g x 1 x h. This is about 4 times higher than the volume / time yield obtained by the process of the corresponding De patent publication.

The degree of purity of the isomaltulose prepared according to the present invention is exactly the same as that of the product prepared according to DE patent publication 217 628.

Example 1 a) Cells inoculated with 10 ml of a sterile solution of 1 part thick Hua (65% dry matter, 92-98% sucrose and the rest of the inoculum) were inoculated with protaminobacter rubrum strain Z 12 (CBS 574 77). other sugars or impurities from sugar beet) and 2 parts of tap water and 0,5 g / l (NH4) 2 ^ Ρ04 (adjusted to pH 7.2 with hydrochloric acid, if necessary). This suspension serves as an inoculum for shaking preculture in 1 liter flasks containing 200 ml of a nutrient solution of the above composition (sterilized for 20 minutes at 121 ° C). After an incubation period of 30 hours at 29 ° C, the contents of 20 flasks (4 L) were transferred to 16 liters of nutrient solution of the above composition in a 30 liter miniature fermentor and fermented at 18 ° C at 20 rpm and 350 rpm with stirring. The conversion of sucrose to isomaltulose was monitored by quantifying the reduction in nutrient solution using Mtille's solution (Zeitschrift Wirt. Zuckerind. Techn. Tf 86, p. 130 and p. 322 (1936) and Zeitschrift Wirt. Zuckerind. Techn. T., 88, p. 280). (1938)). The bacterial count was determined microbiologically. When 9 x 10 bacteria / ml were reached, sucrose had been converted to 100% isomaltulose at a dilution rate of 0.09 / h.

b) According to Example a), an inoculum and preculture of a Protaminobacter rubrum strain were prepared to a scale of 30 liters at a temperature of 29 ° C in each case. Twenty liters of nutrient solution in the growth stage was transferred from a small fermentor to a 300 liter fermentor with 180 liters of nutrient solution (thick juice sucrose-water mixture, dry matter content 25% and purity 96% by dry weight). The aeration ratio of the fermentor was 200 l air / min., The temperature 29 ° C and the agitation speed of 7,64641 rpm. After a seven-hour growth phase, sucrose had been converted 100% to isomaltulose at a dilution rate of 0.09 / h.

c) Cells were inoculated from the inoculum of Protaminobacter rubrum strain Z 12 (CBS 574 77) with 10 ml of a sterile solution containing one part thick juice (65% dry matter) and two parts tap water and 0.5 g / l (NH 4) HPO 2 (if necessary, the pH of the solution was adjusted to 7.2) This suspension was inoculated into a shake preculture in 1 l culture flasks containing 200 ml of a nutrient solution of the above composition (sterilized for 20 minutes at 121 ° C). incubated for 30 hours at 29 ° C, after which the contents of each of the 20 beakers (41) were transferred to 16 liters of a nutrient solution of the above composition in a 30 liter miniature fermentor and fermented at 29 ° C at 20 rpm with stirring at 350 rpm. .

After a 15-hour growth phase at a dilution rate of 0.2 / h, sucrose had been converted 100% to isomaltulose.

d) According to Example a), an inoculum and a preculture of a Protaminobacter rubrum strain were prepared as a shake culture. According to Example b), 0.2 l of inocula (0.1%) were transferred to a fermentor. The fermentation conditions were as in Example b). After a growth period of 22 hours, sucrose had been converted to 90% isomaltulose at a dilution rate of 0.25 / h. When the removed nutrient solution was then transferred to a centrifuge, the reaction continued without further aeration, and thus sucrose could no longer be detected by high pressure liquid chromatography in the nutrient solutions separated from the bacteria.

e) To a 3000 liter fermenter containing 1800 l of nutrient solution (thick juice-sucrose-water mixture, 25% dry matter, purity 98%), 200 l of inoculum obtained from the fermentation according to Example 2c in a 20 liter culture flask were added and fermentation was performed. . Once this fermentation is complete (after about 20 hours), the nutrient solution can be used as an inoculum for the next fermentation (3000 L). The fermentation temperature was 30 ° C. The aeration ratio was 0.4 parts by volume of air / part by volume of nutrient solution x minutes and the stirring speed was 140 rpm. Fermentation was monitored by removal with CCl 2 content. When a CCl 2 content in the exhaust air of 2.6% was reached, a dilution rate of 0.13 / h was used, so that no sucrose but only isomaltulose could be detected in the effluent solution.

Example 2 a) Cells were inoculated from 10 ml of Serratia plymuthica strain (ATCC 15,928) with 10 ml of thick juice solution (dry matter content 25%, purity 96%, added 0.5 g / l (NH 4) 2 O 4). This suspension served as an inoculum for the shaking preculture in a 1 liter flask containing 200 ml of a sterile nutrient solution of similar composition. After an incubation period of thirty hours at 29 ° C, the contents of 20 flasks (4 L) were transferred to 16 liters of nutrient solution of the above composition in a 30 L miniature fermentor and fermented at 29 ° C at 20 L / min and 350 rpm. Under these conditions, the Serratia plymuthica strain increases. When

Q

had reached a bacterial count of 4 x 10 bacteria / ml, sucrose had been converted to 100% isomaltulose at a dilution rate of 0.14 / h.

b) An inoculum and preculture of the Serratia plymuthica strain were prepared according to Example 2a) with a purity of the nutrient solution of 98%. Under the same fermentation conditions as in Example 2a), sucrose was converted to 100% isomaltulose at a dilution rate of 0.09 / h.