Note: Descriptions are shown in the official language in which they were submitted.
2~8~
The present invention is concerned with a process
for the removal of cyclodextrin residues :Erom fats and
oils from which complexable, undesired su~stances, for
example sterols, especially cholesterol, ~ree fatty
acids, vitamins, colouring materials and other
impurities have been removed with the help of cyclo-
dextrin and which still have a certain residual content
of cyclodextrin.
It is known that increased cholesterol levels in
the blood serum of humans represent an increased risk
~ac~or for arteriosclerosis and for coronary heart
disease. For this reason, the endeavours of the food-
stuff industry are markedly to reduce the content of
cholesterol and cholesterol esters in faL-rich food-
stuffs of animal origin, an important problem therebybeing substantially to maintain the sensory and
nutritional-physiological properties of the foodstuffs.
A relatively gentle process for the removal of
cholesterol derivatives is the complexing of these
substances with ~-cyclodextrin and separation of the
complex formed.
According to EP-A 0 256 911, a liquefied fat mass
is treated with a solid cyclodextrin, the cholesterol
value being said to be reduced by up to 80%. However,
for this purpose~ a repeated cyclodextrin treatment is
necessary.
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From W. Schlenk and V.M. Sand (J.S.C.S., 84,
2312/1961), it i5 Icnown to complex free i~atty acids
with cyclodextrin.
In the case of these processes 9 it is an important
problem that a certain amount of the dissolved cyclo-
dextrin remains behind in the fats and oils when the
previously known ~ethods are employed for the separation
thereof. Normally, cyclodextrins (, ~- and ~)~ which
are annular oligosaccharides consisting of 6 to 8
glucose units, are not toxicological. Thus, tests for
the acute toxicity in mice and rats have also given no
indication of a toxic effect in the case of oral
administration. Nevertheless, in many countries, cyclo-
dextrins have hitherto not been permitted to be used as
'~ood additives".
Therefore, it is an object of the present
invention to provide a process for the removal of cyclo-
dextrin residues from fats and oils which, without great
technical expense, makes possible a substantial and
selective removal of cyclodex-trin from the treated
starting materials.
Thus, according to the present invention, there
is provided a process for the removal of cyclodextrin
residues from fats and oils, wherein fats and oils are
emulsified with water and the cyclodextrin is broken
down enzymatically with the help of at least one a-
amylase selected from hog pancreatic amylase and the
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a-amyla~3es formed by micro-organisms of the group
Aspergillus niger, Aspergillus oryzae, Bacillus polymyxa
and Bacillus coagulans, as well as Flavobacterium and/or
preferably of a CTGase selected from the CTGases
formed by bacteria of the group Bacillus, Klebsiella,
Micrococcus and alkalophilic bacteria.
Surprisingly, we have shown that, in this way,
a practically complete breakdown of the cyclodextrin
in fats and oils can be achieved in an economically
acceptable way. This is surprising because cyclo-
dextrins have been described as being potent inhibitors
of the a- and ~-amylases (see R.J. Weselake and
R.D. Hill, Cereal Chem., 60, 98/1983) and are substant-
ially resistant to most - and ~-amylases. We have now
found that a few types of -amylase, for example those
from ~acillus polymyxa and Aspergillus oryzae, as well
as hog pancreatic amylase~ are admittedly able to
hydrolyse cyclodextrins but with low veloclties. There-
fore, i~ was especially surprising and not foreseeable
that this activity would suffice to break down the
cyclodextrin in fat and oil emulsions relatively quickly
and completely.
In the process according to the present invention,
the starting material, consisting of fats and oils of
animal or vegetable origin, for example butter fat, hog
and ~eef fat, goose fat, fish oils, sunflower oils and
the like, which, because of a treatment with cyclo-
_ 4 _ ~5 ~ 03 ~
dextrin, still have a residual content of cyclodextrinsof, for example, 0.001 to 1.0% by weight, are subjected
to an enzymatic treatment.
PreEerably, there are thereby used ~-amylases
selecte~ from hog pancreatic amylase and the a-amylases
formed by micro-organisms of the group Aspergillus
niger, Aspergillus oryzae, ~acillus polymyxa and
Bacillus coagulans, as well as Flavobacterium~ From
the group of the CTGases (cyclodextrin trans-glycosylases
= EC 2.4.1.19), those CTGases formed by bacteria of the
group Bacillus (for example Bacillus macerans, Bacillus
megateriuTn, Bacillus s~earothermophilus, Bacillus
circulans and Bacillus ohbensis), Klebsiella (for
example pneumoniae), Micrococcus (for example varians)
and alkalophilic bacteria, for example Nos. 38-2 and
17-1, are especially preferred. The mentioned enzymes
can also be expressed from host cells of other origin
by methods of genetic manipulation. These ~-amylases
and CTGases make possible a practically complete break-
down of the cyclodeY~trin. The necessary amount ofenzyme thereby depends essentially upon the initial
content of cyclodextrin in the fat or oil and, in the
case of ~-amylase, is usually from 10 to 500 FAU per g
of cyclodextrin to be removed (1 FAU = one fungal a-
amylase unit breaks down 5.2 g of starch in one hourunder standard conditions (subs-trate: soluble starch,
incubation time 7 to 20 minutes, temperature 37C,
_ 5 _ 2 ~ 8 ~ ~ ~
p}l = 4.7)). In the case of the CTGases, these are
preferably used in an amount of 0.5 to 20 U per g of
cyclodextrin to be removed (1 unit = conversion of
1 ~mol of substrate per minute). It is also possible
to use larger amounts of enzyme. However~ in general
this is uneconomical because an improved effect is not
obtained. According to a preferred embodiment of the
process according to the present invention, mixtures of
a-amylase and CTGase are used.
It is important for the present invention to use
the fat or oil in the form of an oil-water emulsion.
For this purpose, the starting material~ possibly after
liquefaction, is emulsified in water, preferably 0.1 to
5 kg of water thereby being added per kg of fat or oil.
The water can already contain the a-amylase and/or
CTGase. However, the enzyme can also be added to the
emulsion after the formation thereof. Before -the
addition of the enzyme, the pH value of the water or
of the emulsion is preferably adjusted to one which is
favourable for the enzyme used and especially -to one
which is optimal for the enzyme used. The adjustment
of the pH value is preferably carried out with buffer
substances and especially edible substances, for
example citric acid, lactic acid and salts thereof.
Co-factors, for example divalent ions, such as calciu,n
or magnesium ions, can possibly be added in trace
amounts -to the emulsion. Furthermore, acceptors, for
~ 6 ~ 8038
example glucose, can also be admixed with the CTGases.
The treatment conditions, for example temperature
and period of time, can be varied within certain limits
but the fats and oils should be present in the liquid
phase, i.e. the enzymatic decomposition should be
carried out at a temperature between the melting point
of the fat or oil in question and 70C. The temperature
for the enzymatic decomposition is preferably from 25 to
55C, in which case treatment times of from 1 to 5 hours
are usual.
With the help of the process according to the
present invention, it is possible to carry out a
substantial and selective removal of the cyclodextrin
from the treated fat or oil in a technically especially
simple manner, the content of residual cyclodextrin
thereby being below a limit of detection of < 10 ppm~
The following Examples are given for the purpose
of illustrating the present inven~ion:
Example 1.
1 kg of fish oil which has been pretreated with
~-cyclodextrin, with a residual content of ~-cyclo-
dextrin of 150 ppm, was mixed with 1 kg of water and
emulsified at 40C. The pH value of the emulsion was
adjusted with citric acid to 5.5. Subsequently, 50 FAU
of an ~-amylase mixture from Aspergillus oryzae were
added thereto in the form of the commercially available
product Fungamyl 800 (Novo). After incubation for
_ 7 _ ~0 ~ ~ 0
2 ~ours at 40C, ~-cyclodextrin could no longer be
detec~ed (detection limit 10 ppm).
Ex ple 2.
1 kg of a butter oil pretreated with ~-cyclodextrin
with a residual content of ~-cyclodextrin of 2500 ppm
was emulsified in 1 kg of water at 50C. The pH value
of the emulsion was adjusted to 6.2 with citric acid
and incubated for 2 hours at 50C with 250 FAU of an
a-amylase from Bacillus coagulans (non-purified prepar-
ation). After this treatment, ~-cyclodextrin could no
longer be detected (detection limit 10 ppm).
Example 3.
1 kg of bee~ tallow~with a resldual content of
a-cyclodextrin of 200 ppm was emulsified in a 5 mmol/l
solution of calcium sulphate at 40C and the pH value
was adjusted to 6.5 with citric acid. Subsequently,
5 g of glucose and 20 U of a CTGase from Bacillus
macerans were added to the emulsion. After incubation
at 40C for 2.S hours~ a-cyclodextrin could no longer
be detected (detection limit 10 ppm).
