DE2741197A1 - Continuous sucrose conversion to iso:maltulose - during continuous fermentation of iso:maltulose-forming microorganisms with addn. of sucrose-contg. solns. - Google Patents

Abstract

Sucrose-contg. solns. are added continuously to a medium in which isomaltulose-forming microorganisms (I) are cultivated continuously. Cultivation si continued in a standard manner. After sucrose conversion, the soln. is separated from the microorganism mass, purified by a standard method and/or crystallised. Isomaltulose is an intermediate for the prepn. of glucopyranosido-1,6-mannitol and glucopyranosido-1-,6-sorbitol. Both cpds. may be used as sugar substitutes. The bacterial mass separated may be used as cattle fodder. The cultivation of the microorganisms and sucrose conversion to isomaltulose take place simultaneously. The bacteria grow quickly on reaching the steady state. 90-100% conversion to isomaltulose is achieved. Sucrose-contg. plant juices, cheaper than pure sucrose-contg. solns., may be used. Bacterial growth and conversion can be conrolled by only one critical parameter, e.g. CO2 content in waste air, O2 partial pressure in culture soln. and/or conversion rate. Fructose and glucose are formed during conversion to isomaltulose, obviating the need for adding carbohydrate sources.

Description

Process for the continuous fermentation of microorganisms

organisms with simultaneous conversion of sucrose into isomaltulose The invention relates to a process for the continuous fermentation of Microorganisms with simultaneous conversion of sucrose into isomaltulose (palatinose, α-D-glucopyranosido-1,6-fructose).

Isomaltulose is an intermediate product in the production of glucopyranosido-1,6-mannitol (DT-OS 2 520 173) and glucopyranosido-1,6-sorbitol (isomaltitol, DT-PS 2 217 628). Both Substances can be used as sugar substitutes.

According to German Patent 1,049,800 it is known that sucrose is enzymatically converted into isomaltulose. The enzymes to do this are microbial Origin. In addition to Protaminobacter rubrum, there are other bacteria such as Erwinia carotovora, Serratia marcescens, Serratia plymuthica and Leuconostoc mesentheroides to this one Relocation enabled (S. Schmidt-Berg-Lorenz, W. Mauch, magazine for the sugar industry 14: 625-627 (1964); F.H.

Stodola 126th Meeting Amer. Chem. Soc., Sept. 1954, Abstracts of Papers, P. 5 D; W. Mauch, S. Schmidt-Berg-Lorenz, magazine f.d. Sugar industry 14, 309 - 315 and 375-383 (1964)).

Furthermore, according to German patent specification 2 217 628, it is known that the enzymatic conversion of sucrose into isomaltulose with a 15-40% strength Solution under vigorous stirring and aerobic conditions at 20 - 370C in batches or can be carried out continuously. After the enzymatic reaction has ended According to the above patent specification, the bacterial suspension is separated off using a separator and reused up to six times. With the continuous implementation of sucrose According to the same patent specification, the bacteria are placed on a special nutrient solution grown and continuously withdrawn. The implementation then takes place in a second Kettle cascade system in which bacterial culture and syrup from sugar production or another sugary solution.

The separate cultivation of the microorganisms from the enzymatic conversion the sucrose is unsatisfactory for various reasons: a) The microorganisms are attracted in their own medium, which causes increased costs.

b) The culture solution and the fermenter must be independent of the Reactor boiler system in which the enzymatic rearrangement takes place, sterilized and are kept sterile.

c) The cultivation of microorganisms in a nutrient solution that is different of the later sales solution, there is a risk that the microorganisms during the fermentation is selected according to criteria other than the maximum conversion rate will.

d) Separating the microorganisms and reusing them for further enzymatic conversions are on an industrial scale with a high risk of infection tied together.

It has now been found that the cultivation of isomaltulose-forming Microorganisms with simultaneous conversion of sucrose into isomaltulose continuously can be carried out.

Among isomaltulose-forming microorganisms were mainly Protaminobacter rubrum, also Serratia plymuthica, Serratia marcescens, Erwinia carotovora and Leuconostoc mesentheroides understood.

It was surprising and not expected to be isomaltulose-forming Microorganisms, e.g. Protaminobacter rubrum, in plant juices containing sucrose, which, for example, arise as an intermediate product in the production of cane and beet sugar and hence cheaper than pure sucrose solution while growing at the same time Convert sucrose into isomaltulose. According to the invention, this is the growth of the organisms coupled with the conversion rate in such a way that the entire process, i.e.

Growth and implementation, only via a critical parameter, such as C02 content in the exhaust air, oxygen partial pressure in the culture solution or else can be controlled via the conversion rate.

Another advantage of the procedure is the following: During the Rearrangement to isomaltulose creates small amounts of fructose and glucose, which the Microorganisms serve as carbohydrates for their metabolism. There must be no other sources of carbohydrate for growth added to the process will. At the same time, these undesirable by-products are largely broken down.

In continuous isomaltulose fermentation one uses as Starting solution thin juice / thick juice and / or thin juice / clarification mixtures from a sugar factory with a dry matter content of 5 - 30%, preferably 20 - 27%. The sucrose content, based on dry weight, is 90-98%, preferably 94-96%.

In order to achieve optimal growth of the bacterial culture, be Phosphate ions in the form of salts such as (NH4) 2HP04 or K2HPO4 in concentrations from 0.05 to 1 g / l, preferably from 0.1 to 0.5 g / l, added. Usually included the intermediate products of sugar production still have a residual CaO content after the juice purification from 40 - 150 mg to 100 g DM. This residual Ca0 content becomes 3-the addition of Po4 precipitated as calcium phosphate and thus part of the added phosphate goes for the cell proliferation is lost.

Therefore, the sugar solution to be fermented is advantageously used Softened via a base exchanger, which 3-lowers the addition of PO4 ions can be.

The nutrient solution was sterilized batchwise or continuously; pH correction is not necessary if the pH value of the sugar solution used is between 7.5 and 8.5.

The fermenter is grown with 0.1-10% inoculum in the same Nutrient solution with Erlenmeyer flask on the shaker, inoculated and with a Temperature of 18 - 329C, preferably at 300C, with an aeration rate of 0.2 - 1.0 Volume of air per volume of nutrient solution times minute (vvm), preferably 0.4 vvm, incubated with sufficient agitation.

9 After reaching a sufficient cell density, e.g. 109 germs per ml the fermentation is carried out continuously, i.e.

Equal proportions of the culture solution are removed per unit of time and fresh, sterile, uninoculated nutrient solution added from a storage tank. At an optimal dilution rate between 0.05 and 0.3 hours 1, preferably is at 0.2 hours 1, the bacteria are not washed out. The bacteria grow so fast in the "steady state" that a 90-100% conversion into isomaltulose is achieved. A stabilization of the pH value during the reaction is in the Rule. not mandatory.

The rate of dilution and thus the outflow of the culture solution from the continuous fermentation is carried out, among other things, with sterilizable oxygen electrodes via the oxygen partial pressure in the culture solution or via the CO2 content in the exhaust air or controlled by other physiological parameters. One more way The control of the continuous fermentation is the control of the conversion rate of sucrose in isomaltulose by determining the reducing power of the fermentation solution. (As is well known, the reducing power of isomaltulose is 52% of that of glucose, while sucrose has no reducing properties.) A second possibility the control of the conversion rate is the quantitative detection of isomaltulose in addition to sucrose with the help of high pressure liquid chromatography.

The nutrient solution withdrawn from the fermenter is then from separated the microorganisms e.g. with a separator and further worked up. The thrown off bacterial mass is a second time with water Floated, centrifuged and can be used as fodder, among other things.

If the culture solution withdrawn from the fermenter is only e.g.

to 90 z is implemented is by the microorganisms without renewed Supply of oxygen in the pipe system to the separator ensures complete implementation carried out.

Example 1 a) From a vaccination of the Protaminobacter rubrum strain cells are treated with 10 ml of a sterile mixture of one part thick juice (TS = 65 %) and two parts of tap water plus 0.5 g / l (NH4) 2HP04 (if necessary with HCl adjusted to pH 7.2) washed away. This suspension is used as an inoculum for the shaking machine preculture in 1 1 flasks with 200 ml of nutrient solution of the above composition (Sterilization 20 min. At 1210C). After an incubation period of 30 hours at 290C each with 20 flasks (4 1) 16 1 nutrient solution of the above composition in a 30-liter small fermenter inoculated and at 180C with 20 1 air per minute and a stirring speed of Fermented at 350 rpm.

The conversion of the sucrose into isomaltulose is through the quantitative Determination of the reduction behavior of the nutrient solution with the help of Müller's solution (Zeitschrift Wirt. Zuckerind. Techn. T. 86, p. 130 and p. 322 (1936) and magazine Host. Sugar ind. Techn. T. 88, p. 280 (1938). The germ count becomes microbiological definitely 9. After reaching 1 109 germs / ml, the dilution rate is increased every hour 100% conversion achieved after 0.09 h.

b) Inoculation material and preculture from the Protaminobacter rubrum strain are made accordingly Example a) produced up to a 30-liter scale, but in each case at temperatures Von 290C. 20 1 growing nutrient solution from the small fermenter serve as inoculation material for a 300 l fermenter with 180 l nutrient solution (thick juice-sucrose-water mixture with 25% dry matter content and a purity of 96% on TS). The fermenter runs at a ventilation rate of 200 1 air / min., One temperature of 290C and a stirring speed of 200 rpm.

After a growth phase of 7 hours, use a dilution rate of 0, O9h 1 achieved a 100% conversion rate.

c) Inoculum and preculture from the Protaminobacter rubrum strain are made accordingly Example a) produced up to shaking culture. With 2 1 inoculum from the shaker (= 1%) a fermenter is inoculated according to b), fermentation conditions according to Example b). After the growth phase of 15 hours, a dilution rate is used achieved a 100% conversion rate of 0.2 h 1.

d) Inoculum and preculture of the Protaminobacter rubrum strain are made accordingly Example a) produced up to shaking culture. With 0.2 1 inoculum a fermenter is made inoculated according to example b) (0.1%), fermentation conditions accordingly Example b). After a growth phase of 23 hours, a dilution rate is used A 90% conversion was achieved in 0.25 h 1. During the subsequent transport of the withdrawn Nutrient solution to the separator, the implementation continues without additional ventilation, so that in the nutrient solutions separated from the bacteria no sucrose with the help liquid high pressure chromatography is more detectable.

e) A 3000-1 fermenter is filled with 1800 1 nutrient solution (thick juice-sucrose-water mixture, 25 z dry matter content, Purity 98%) and 200 1 inoculum drove another 2000-1 fermentation. The temperature is 30 ° C.

The ventilation rate 0.4 vvm, the stirring speed 140 rpm.

The fermentation is tracked via the CO 2 content in the exhaust air. After reaching 2.6% CO in the exhaust air, a dilution rate of 0.13 h driven, with no sucrose in the draining culture solution, but only isomaltulose is still detected.

Example 2 a) From a vaccination of the Serratla plymuthica strain (ATCC 15 928) cells with 10 ml of a syrup solution (25% dry matter content, 96% Purity, 0.5 g / l (NH4) 2HP04 additive). This suspension is used as an inoculation material for a shaking machine preculture in a 1 l flask with 200 ml of sterile nutrient solution corresponding ~ composition. After an incubation time of 30 hours at 290C each with 20 flasks (4 1) 16 1 nutrient solution of the above composition in a 30 l small fermenter inoculated and at 29 0C with 20 1 air / min. and a stirring speed of 350 rpm fermented. After reaching a certain 9 germ count, e.g. 4 x 109 germs / ml, 100% conversion is achieved with an hourly dilution rate of 0.14 h 1 achieved.

b) Inoculation material and preculture from the Serratia plymuthica strain are made accordingly Example 2a) produced, but with a nutrient solution of 98% purity. at same fermentation conditions as listed under 2a), with an hourly Dilution rate of 0.9 h 1 achieved 100% conversion.

Claims (1)

Method for the continuous fermentation of microorganisms with simultaneous conversion of sucrose into isomaltulose, characterized in that one is a medium in which isomaltulose-producing bacteria are continuously cultured are, continuously adding sucrose-containing solutions, the cultivation in itself in a known manner, after the sucrose has been converted, the solution of the bacterial mass is carried out separates and purifies in a manner known per se and / or brings about crystallization.