Note: Descriptions are shown in the official language in which they were submitted.
The invention relates to a process for the continuous
fermentation of micro-organisms with simultaneous conversion
o~ sucrose into isomaltulose (palat:inose, a-D-glucopyranosido
1,6-fructose)~
Isomal-tulose is an intermed:iate product for the prepara-
tion of glucopyranosido~ -mannitol (DT-OS (German Published
Specification) 2,520,173) and glucopyranosido-1,6-sorbitol
(isomaltitol, German Patent Specification 2,217,628). Both
substances can be employed as sugar substitutes.
It is known, according to German Patent Specification
1,049,800, that sucrose can be enzymatically converted into
isomaltulose. The enzymes for this are of microbial origin.
In addition to Protaminobacter rubrum, fur-ther bacteria, such
as Erwinia carotovora, Serra-tia marcescens, Serratia plymuthica
and Leuconostoc mesentheroides, are capable of effec-ting this
rearrangement (S.Schmidt-Berg-Lorenz, W. Mauch, Zeitschrift f~r
die Zuckerindustrie 14, 625 - 627 (1964); F.H. Stodola 126th
Meeting Amer. Chem. Soc., September 1954, Abstracts of Papers,
page 5 D; -and W. Mauch, S.Schmidt-Berg-Lorenz, Zeitschri~t f.d.
Zuckerin~us-trie 14, 309 - 315 and 375 - 383 (1964-)).
It is also known, according to German Patent Specifica-
-tion 2,2179628, that the enzymatic conversion of sucrose into
isomaltulose can be carried out batchwise or continuously using
- a 15-40% strength solution, whilst stirring vigorously and
under aerobic conditions at 20 - 37C. According to the
above patent speci~ication, after -the en~yma-tic reaction has
ended, the bacteria suspension is separated off via a separator
and re-used up to six times. According to the same patent
specification~ in the continuous conversion o~ sucrose -the
. bacteria are cultured in a special nutrient solution and are
continuously removed. The conversion then takes place in a
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g~ ~
second kettle cascade system, in which the bacteria culture and thick juice
from sugar manufacture or another sugar-containing solution are kept.
However, the division of process in growth of microorgani.sms and
enzymatic conversion of the sucrose is unsatisfactory for various reasons:
a) the microorganisms are grown in a special medium which gives rise to in-
creased costs; b) the culture solution and also the fermenter must be ster-
ilised, and kept under sterile conditions, independently of the reactor
kettle system in which the enzymatic rearrangement takes place; c) cultur-
ing the microorganisms in a nutrient solution which differs from the later
solution for the conversion involves the danger for the microorganisms being
selected, during the fermentation, by criteria other than that of the maximum
rate of conversion; and d) separating off the microorganisms ancl re-using
them for further enzymatic conversions is associated with a high danger of
inEection on an industrial scale.
It has now been found that the culture of isomaltulose-formi.ng
microorganisms with simultaneous conversion of sucrose into isomaltulose can
be carried out continuously.
Accordingly the present invention provides a process for the con-
tinuous fermentation of microorganisms with simultaneous conversion of sucrose
into isomaltulose, in which a sucrose-containing solution is continuously
added to a medium in which an isomaltulose-forming microorganism is being
continuously cultured, culturing is carriecl out and a:Eker the conversion of
the sucrose, the solution is separated off from the microorganism mass and
purified and/or made to crystallise.
By isomaltulose-forming microorganisms there were understood here,
above all, Protaminobacter rubrum, and in addition Serratia plymuthica,
Serratia marcescens, Erwinia carotovora and Leuconostoc mesentheroides.
It was surprising, and not to be expected, that during growth,
isomaltulose-forming microorganisms, for example Protaminobacter rubrum,
(~BS 57~.77) in sucrose-containing vegetable juices which are obtained, for
example~ as an intermediate product in the production of cane sugar and beet
sugar and thus are cheaper than pure saccharose solution, at ~he same
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time convert sucrose into isomaltulose. According to the
invention, the grow-th of the organisms is linked with the rate
of conversion such that the entire process, that is -to say
growth and conversion, can be monitored via only one critical
parameter, such as, for example, the content of C02 in the
waste air or the partial pressure of oxygen in the culture
solution, or via the rate of conversion.
A further advantage of the process is the following:
small amounts of ~ructose and glucose which are used by the
micro-organisms as carbohydrates for their metabolism are
formed during the rearrangement to isomaltulose. In the
process, no further carbohydrate sources need be added for the
growth, At the same time, substantial degradation of these
undesired by-products is achieved.
Thin juice/thick juice mixtures and/or thin juice/clear
liquid mixtures from a sugar factory which have a dry substances
content of 5 - 30%, preferably of 20 - 27%, are used as the
starting solution in the continuous isomaltulose fermentationO
The sucrose content, relative to dry weight, is 90 - 98%,
pre~erabl~ 94 - 96%.
In order to obtain optimum growth of the bacterial
culture~ phosphate ions in the form of salts, such as, for
example, (NH4)2HP04 or K2HP04, are added in concentrations of
0.05 - 1 g/l, preferably of 0.1 - 0.5 g/1. After -the
puriflcation of the juice, the intermediate products from the
manufacture of sugar usually still contain a residual content
o~ CaO of 40 - 150 mg per 100 g of dry substances This
residual content of CaO is precipitated as calcium phosphate by
adding Po43~, and some of the phosphate added for cell multi-
plication is thus lost
The sugar solution used for the fermentation is there-
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.: .,. .... :: ..... .. : .. , .... .. . . . - . . .. .
fore advantageously sof-tened via a base exchanger, by which
means the amount of P043 ions added can be lowered.
The nutrien-t solution was sterilisecl batchwise or
continuously; it is not necessary t;o correct -the pH if the
pH value of the sugar solution used is between 7.5 and 80 5.
The fermenter is inoculated with 0.1 - 10% of inoculum,
grown in the same nutrient solu-tion using conical flasks on a
shaking machine, and the mixture is incubated at a temperature
of 18 - 32C, preferably at 30C, with an aeration rate of
0.2 - 1.0 volume of air per volume of nutrient solution times
minutes (vvm), preferably o , 4 vvm, whilst stirring adequately.
After an adequate cell densi-ty has been achieved, ~or
example 109 germs per ml, the ~ermentation is carried out con-
tinuously, -that is -to say the culture solution is removed and
fresh, sterile, non-inoculated nutrient solution Prom a reser-
voir is added in equal por-tions per unit time At an
optimum dilution rate, which is between 0.05 and 0.3 hour 1
and is preferably 0 2 hour 1, -the bac-teria cannot be washed
out, In the steady state the bacteria grow so rapidly -that
90 - 100% conversion into isomaltulose is achieve~d. As a
rule, it is not necessary to stabilise the pH value during the
conversion.
The dilution rate and thus the ~low of the culture
solution out of the continuous fermentation is monitored, inter
alia, via the partial pressure of oxygen in -the culture solu-
tion, using oxygen electrodes which can be sterilised, or via
the C0z content in the waste aîr or via further physiological
parameters. A fur-ther possibility of monitoring the con-
tinuous fermentation is to monitor the rate o~ conversion of
sucrose into isomaltulose by cletermining -the reducing power
of the fermentation solution. (As is known, the reducing
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. . . . .
power of isomaltulose is 52% of tha-t of glucose, whilst sucrose
has no reducing properties,) A second possibility o~ --
monitoring the rate of conversion is the quantitative deter-
mination of isomal-tulose in addition to sucrose with the aid of
high pressure liquid chroma-tography~,
The nutrient solution removed from the fermenter is
then separated o~f from the micro-organisms~ for example using
a separator, and further worked up. The bacteria mass which
has been centrifuged off is suspended in water and centrifuged
a second time and can be used, inter alia, as fodder.
If,the culture solution removed from the fermenter is
converted, for example, to the exten-t of only 90%, complete
conversion is carried out by the micro~organisms in the line
s~stem to the separator wi-thout renewed addition of oxygen.
~3~æa~
a) Cells from a transinoculation of the strain Protamino-
bacter rubrum Z 12 (CSB 574.77) are suspended with 10 ml of a
sterile mixture of one part of thick juice (dry substances =
65%) and two parts of tapwater plus 0.5 g/l of (NHL~)2HP04 (if
necessary adjusted to pH 7.2 wi-th HCl), This suspension is
used as the inoculum for -the shaking machine precul-ture in 1 1
flasks with 200 ml of nutrien-t solu-tion o~ -the above composi-
tion (sterilisation 20 minutes at 121C). After an incuba-
tion time of 30 hours at 29C, 16 1 of nutrient solution of the
above composition are inoculated with each of 20 flasks (4 1)
in a ~0 1 small fermenter and fermentation is carrled out at
18C with 20 1 of air per minute and a stirring speed of 350
revolutions per minute. The conversion of sucrose into
isomaltulose is followed by -the quantitative determination of
the reducing properties of the nutrient solut;ion with the aid
of M~ller's solution (Zeitschrift ~irt. Zuckerind. Tech. part
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86, page 130 and page 322 (1936) and Zeitschrift Wirt. Zucker-
ind. Techn. part 88, page 280 (1938). The number of germs
is determined microbiologically. Af-ter an amount o~ 1 . 109
germs/ml has been reached, 100% conversion is achieved with
an hourly dilution rate of 0.09 hour 1,
b) The inoculum and preculture of the s-train Protamino-
bacter rubrum are prepared according to Example a) up to the
30 1 scale, but in each case at temperatures of 29C. 20 1
of nutrient solution, in which growth has taken place, from the
small fermenter are used as the inoculum for a 300 1 fermenter
containing 180 1 of nutrient solution (thick li~uid/sucrose/
water mixture with a dr~ substances content of 25% and a purity
of 96%, relative to the dry substances). The ferm0nter
operates at an aeration rate of 200 1 of air/minute, a tempera-
ture of 29C and a stirring speed of 200 revolutions per
minute.
After a gro~th phase of 7 hours, a con~ersion rate of100% is achieved with a dilution rate of 0.09 hour 1,
c) The inoculum and precul-ture of the strain Protamino-
bacter rubrum are prepared according to Example a) up to theshaken culture. A fermenter according to b) is inoculated
with 2 1 of inoculum from the shaking machine (- 1%); the
fermentation conditions correspond to Example b). After
the growth phase of 15 hours, a conversion rate of 100% is
achieved with a dilution rate o~ 0.2 hour 1,
d) The inoculum and preculture of the strain Protamino-
bacter rubrum are prepare~ according to Example a) up to the
shaken culture. A fermenter according to Example b) is
inoculated with 0.2 1 of inoculum (0.1%); the fermon-tation
conditions correspond to Example b). After a growth phase
of 23 hours, 90% conversion is achieved with a d:ilution rate
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of 0, 25 hour 1~ When the nutrient solution removed is sub~ --
seauently transported to the separator, the conversion con-
tinues without additional aeration, so that no further sucrose
can be detected, with the aid of high pressure liquid chromato-
5 graphy, in the nu-trient solutions separated off from the
bacteria.
e) A 3000 1 fermenter is operated with 1,8Q0 1 of nutrient
solution (thick liquid/sucrose/water mixture7 25% dry substances
content, purity 98%) and 200 1 Of inoculum ~rom a further 2,000 1
fermentatïon. The te~perature is 30C. The aeration rate 0.4 vvm,the
stirring speed 140 revolutions per minute. The fermentation
is followed via the content of C02 in the waste air. After
2, 6% of C02 has been reached in the was-te air, the fermenter
is operated with a dilution rate of 0.13 hour 1, whereupon no
sucrose but only isomal-tulose is detected in the outflowing
culture solution.
Exam~le 2
a) Cells from a transinocula-tion of the strain Serratia
plymuthica (ATCC 15,928) are suspended in 10 ml of a thick
juice solution (25% of dry substances content, 96Q~o purity,
0.5 g/l of (NH4)2HP04 added), This suspension is used as
the inoculum for a shaking machine preculture in a 1 1 flask
containing 200 ml of sterile nutrient solution of appropriate
composition, After an incubation time of 30 hours at 29C,
16 1 of nutrient solution of the above composition are inocula-
ted-with each of 20 flasks (4 1) in a 30 1 small fermenter and
are fermented at 29C with 20 1 of air/minute and a stirring
speed o~ 350 revolutions per minute~ After a certain number
of germs has bèen reached, for example 4 x 109 germs/ml, 100%
conversion is achieved with an hourly dilution rate of 0.14
hour~l .-
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" ' .. . '
b) The inoculum and preculture of the strain Serratia
plymuthica are prepared according -to Example 2a), bu-t using
a nutrient solution of 98% purity. Under the same fer-
mentation condi-tions as given under 2a), 100% conversion is
achieved with an hourly dilu-tion rate of 0.09 hour 1,
~ _ g _
. .
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