IE47250B1 - Process for the continuous isomerisation of sucrose to isomaltulose with the aid of micro-organisms - Google Patents

Abstract

1. A process for the continuous isomerisation of sucrose into isomaltulose with the aid of micro-organisms, characterised in that isomaltulose-forming micro-organisms are cultured in a manner known per se in a culture medium having a content of sucrose-containing vegetable juices ; after an adequate cell density has been achieved, culturing is continued in a continuous manner by adding sucrose-containing solutions continuously at a dilution rate between 0.05 and 0.3 per hour ; after the sucrose has been isomerised the solution is separated from the micro-organism mass and purified in a manner known per se and/or made to crystallize.

Description

The invention relates to a process for the continuous isomerisation of sucrose to isomaltulose (palatinose, a-D-glucopyranosido-l,6-fructose) with the aid of micro-organisms.

Isomaltulose is an intermediate product for the preparation of glucopyranosido-l,6-mannitol (German Offenlegungsschrift 2,520,173) and glucopyranosido-1,6sorbitol (isomaltitol, German Patent 2,217,628). Both substances can be employed as sugar substitutes.

It is known, from German Patent Specification 1,049,800, that sucrose is enzymatically converted to isomaltulose. The enzymes used for this purpose are of microbial origin. In addition to Protaminobacter rubrum, other bacteria, such as Erwinia carotovora, Serratia marcescens, Serratia plymuthica and Leuconostoc mesentheroides are capable of this rearrangement (S. Schmidt-Berg-Lorenz and W. Mauch, Zeitschrift fur die Zuckerindustrie, 14, 625-627 (1964); P.H.Stodola, 126th Meeting Amer. Chem. Soc., September 1954, Abstracts of Papers, page 5 D; W. Mauch and S. Schmidt-Berg-Lorenz, Zeitschrift f.d. Zuckerindustrie 14, 309-315 and 375-383 (1964)).

Further, it is known from German Patent Specification 2,217,628 that the enzymatic conversion of sucrose to isomaltulose can be carried out, batchwise or continuously, with a 15-40% strength solution, employing vigorous stirring and aerobic conditions, at 20-37°C. According to the above patent specification, after completion of the enzymatic reaction the bacterial suspension is removed by means of a separator and is reused up to six times. According to the same patent specification, in the continuous reaction of sucrose the bacteria are cultured on a special nutrient solution and taken off continuously. The reaction is then carried out in a second kettle cascade system, in which the - 3 bacterial culture and syrup from sugar manufacture or some other sugar-containing solution are kept.

The culturing of the micro-organisms separately from the enzymatic reaction of the sucrose is, however, unsatisfactory for various reasons: a) The micro-organisms are cultured in a special medium, which entails increased costs. b) The culture solution and also the fermenter must be sterilised, and kept sterile, separately from the reactor kettle system in which the enzymatic rearrangement takes place. c) The culturing of the micro-organisms in a nutrient solution which is different from the subsequent reaction solution entails the danger that the micro-organisms may, during the fermentation, be selected according to different criteria than that of the maximum reaction rate. d) The separating-off of the micro-organisms and the re-use for further enzymatic reactions entails, on the industrial scale, a high risk of infection.

It has now been found that sucrose can be continuously isomerised to isomaltulose if isomaltuloseforming micro-organisms are cultured, in a manner known per se, in a culture medium having a content of sucrosecontaining vegetable juices and, after an adequate cell density has been achieved, are continuously cultured further by adding sucrose-containing solutions continuously at a dilution rate between 0.05 and 0.3 per hour, and, after isomerisation of the sucrose, the solution is separated from the micro-organism mass and is, in a manner known per se, purified and/or made to crystallise.

By isomaltulose-forming micro-organisms there have here been understood, above all, Protaminobacter rubrum, Serratia plymuthica, Serratia maroescens, Erwinia carotovora and Leuconostoc mesentheroides.

It was surprising and not to be expected, that isomaltulose-forming micro-organisms, for example Protaminobacter rubrum (CBS 574.77), in sucrose-containing vegetable juices which arise, for example, as an intermediate product in the production of cane sugar and beet sugar, and are hence cheaper than pure sucrose solution, during growth simultaneously convert sucrose to isomaltulose. Furthermore, according to the invention, the growth of the organism is coupled with the reaction rate in such a way that the total process, that is to say growth and reaction, can he controlled via only one critical parameter, such as, for example, CO^ content in the exit air or partial pressure of oxygen in (sic) the culture solution, or via the reaction rate (sic).

A further advantage of the process is the following: during the rearrangement to isomaltulose, small amounts of fructose and glucose are formed, which serve the micro-organisms as carbohydrates for their metabolism. According to the process, no further sources of carbohydrate need be added for growth.

At the same time, substantial degradation of these undesired by-products is achieved.

In the continuous fermentation of isomaltulose, thin juice/syrup mixtures and/or thin juice/clarifier liquor mixtures from a sugar factory, the mixtures having a solids content of 5-30%, preferably of 20-27%, are used as the starting solution. The sucrose content, based on solids, is 90-98%, preferably 94-96%.

To achieve optimum growth of the bacterial cul25 ture, phosphate ions in the form of salts, such as, for example, or K/iPC/ are added in concentrations of 0.05-1 g/l, preferably of 0.1-0.5 g/l. Normally, the intermediate products of .sugar manufacture still contain, after liquor purification, a residual CaO content of 40-150 mg per 100 g of solids content. This residual CaO content is precipitated, as calcium phosphate, hy the addition of PO^^-, and consequently a part of the added phosphate is lost to cell multiplication purposes.

Hence, the sugar solution employed for ferment35 ation is advantageously softened by passing over a base exchanger, as a result of which the amount of added P0^“ ions can be decreased.

The nutrient solution was sterilised batchwise or continuously; a pH correction is not necessary if - 5 472S0 the pH value of the sugar solution is between 7.5 and 8.5.

The fermenter is inoculated with 0.1-10% of inoculum material, grown in the same nutrient solution with (sic) Erlenmeyer flasks on a shaking machine, and is incubated at a temperature of 18-32°C, preferably at 30°C, employing an aeration rate of 0.2-1.0 volume of air per volume of nutrient solution per minute (wm), preferably 0.4 wm, with adequate stirring.

After an adequate cell density, for example 10 cells per ml, has been reached, the fermentation is carried out continuously, that is to say per unit time equal proportions of culture solution are removed and of fresh, sterile, non-inoculated nutrient solution are added from a stock tank. At an optimum dilution rate, which is between 0.05 and 0.3 hours-1, preferably 0,2 hours-1, washing-out of the bacteria does not occur.

In the steady state the bacteria grow so rapidly that 90-100% conversion to isomaltulose is achieved. Stabilisation of the pH value during the reaction is as a rule not necessary.

The dilution rate, and hence the efflux of the culture solution from the continuous fermentation is controlled, inter alia, with sterilisable oxygen electrodes via the oxygen partial pressure in (sic) the culture solution or via the C02 content in the exit air or via other physiological parameters. A further possible method of control of the continuous fermentation is to check the rate of conversion of sucrose to isomaltulose by determining the reducing power of the fermentation solution. (As is known, the reducing power of isomaltulose is 52% of that of glucose, whilst sucrose possesses no reducing properties). A second possible way of checking the reaction rate is the quantitative detection (sic) of isomaltulose alongside sucrose by means of high pressure liquid chromatography.

The nutrient solution drawn from the fermenter is subsequently separated from the micro-organisms, for example by means of a separator, and is worked up further. 47350 - 6 The bacterial mass v/hich has been centrifuged off is suspended in water for a second time and centrifuged, and can inter alia be used as animal fodder.

If the culture solution drawn from the fermenter has, for exanple, only been 90% converted, the complete conversion is effected by the micro-organisms, without renewed supply of oxygen, in the pipeline system leading to the separator.

Example 1 a) Cells are flushed off a trans-inoculated culture of the strain Protaminobacter rubrum Z 12 (CBS 574.77) by means of 10 ml of a sterile mixture of one part of syrup (solids content = 65%) and two parts of tap water plus 0.5 g/l of (NH^J^HPO^ (if necessary adjusted to pH 7.2 viith HCl). This suspension serves as the inoculum for the shaking machine preculture in 1 litre flasks, using 200 ml of nutrient solution of the above composition (sterilisation for 20 minutes at 121°C). After an incubation time of 30 hours at 29°C, 16 litres of nut20 rient solution of the above composition are inoculated, in a 30 litre small-scale fermenter with, in each case, (the contents) of 20 flasks (4 litres), and are fermented at 18°C with 20 litres of air per minute, at a speed of stirring of 350 rpm. The conversion of the sucrose to isomaltulose is followed by quantitative determination of the reduction characteristics of the nutrient solution, by means of Muller’s solution (Zeitschrift Wirt. Zucker ind. Techn.' T. 86, page 130 and page 322 (1936) and Zeitschrift Wirt. Zuckerind. Techn.

T. 88, page 280 (1938). The cell count is determined microbiologically. When 1 . 10θ germs/ml is reached, 100% conversion is achieved with an hourly dilution rate of 0.09 h~\ b) The inoculum and pre-culture of the strain Protaminobacter rubrum are prepared analogously to Example a) up to the 30 litre scale, but in each case at temperatures of 29°C. 20 litres of nutrient solution which had undergone growth in the small-scale fermenter are used as the inoculum for a 300 litre - 7 fermenter containing 180 litres of nutrient solution (syrup/sucrose/water mixture having a solids content of 25% and a purity of 96%, based on solids content).

The fermenter runs with an aeration rate of 200 litres of air/minute, a temperature of 29°C and a speed of stirring of 200 rpm.

After an initial growth phase of 7 hours, 100% conversion rate is achieved at a dilution rate of 0.09 h-1. c) The inoculum and pre-culture of the strain Protaminobacter rubrum are prepared analogously to Example a) up to the shaken culture. A fermenter is inoculated analogously to b) with 2 litres of inoculum from the shaking machine (= 1%), with fermentation conditions corresponding to Example b). After the initial growth phase of 15 hours, 100% conversion rate is achieved with a dilution rate of 0.2 h_1. d) The inoculum and pre-culture of the strain Protaminobacter rubrum are prepared analogously to Example a) up to the shaken culture. A fermenter is inoculated analogously to b) with 0.2 litres of inoculum, with fermentation conditions corresponding to Example b) (0.1%). After the initial growth phase of 25 hours, 90% conversion rate is achieved with a dilution rate of 0.25 h-\ On subsequent transportation of the drawnoff nutrient solution to the separator, the reaction continues without additional aeration, so that in the nutrient solution separated from the bacteria, sucrose is no longer detectable by means of high pressure liquid chromatography. e) A 3,000 litre fermenter is run with 1,800 litres of nutrient solution (syrup/sucrose/water mixture, 25% solids content, purity 98%) and 200 litres of inoculum from a further 2,000 litre (sic) fermentation. The temperature is 30°C. The aeration rate is 0.4 wm and the speed of stirring 140 rpm. The fermentation is followed via the C02 content in the exit air. When 2,6% of C09 in the exit air is reached, the fermentation -i is run with a dilution rate of 0.13 h , under which conditions no sucrose, but only isomaltulose, is detect4725Q - 8 eel in the effluxing culture solution.

Example 2 a) Cells are flushed off a trans-inoculated culture of the strain Serratia plymuthica (ATCC 15,928) hy means of 10 ml of a syrup solution (25% solids content, 96% purity, 0.5 g/l of (ΝΗ^)2ΗΡ0^ added). This suspension serves as the inoculum for a shaking machine preculture in a 1 litre flask containing 200 ml of sterile nutrient solution of corresponding composition. After an incubation time of 30 hours at 29°C, 16 litres of nutrient solution of the above composition are inoculated in a 30 litre small-scale fermenter with, in each case, (the contents) of 20 flasks (4 litres), and are fermented at 29°C with 20 litres of air per minute, at a speed of stirring of 350 rpm. After reaching a Q particular cell count, for example 4 x 10 cells/al, 100% conversion is achieved with an hourly dilution rate cf 0.14 h-^ (sic). b) The inoculum and pre-culture of the strain Serratia plymuthica are prepared analogouly to Example 2a), but with a nutrient solution of 98% purity.

Under the same fermentation conditions as mentioned under 2a), '100% conversion is achieved with an hourly dilution rate of 0.09 h-1 (sic).

Claims (4)

1. CLAIMS:1. A process for the continuous isomerisation of sucrose into isomaltulose with the aid of micro-organisms, characterised in that isomaltulose-forming micro-organisms are cultured in a 5 manner known per se in a culture medium having a content of sucrose-containing vegetable juices; after an adequate cell density has been achieved, culturing is continued in a continuous manner by adding sucrose-containing solutions continuously at a dilution rate between 0.05 and 0.3 per hour; 10 after the sucrose has been isomerised the solution is separated from the micro-organism mass and purified in a manner known per se and/or made to crystallize.

2. A process according to claim 1, characterised in that it is conducted by culturing Protaminobacter rubrum Z 12 (CBS 574.77). 15

3. A process according to claim 1, when carried out substantially as described in any one of Examples 1(a) to (e) and Example 2(a) and (b).

4. Isomaltulose when produced by the process of any of the foregoing claims. F.R. KELLY & CO. AGENTS FOR THE APPLICANTS.