Note: Descriptions are shown in the official language in which they were submitted.
~3~7~
BAC~CGROUND OF 'rho INVENTION
Naturally occurring carbohydrates in the form oE starch are
renewable sources of commerically important sugars ineluding the mono-
saccharido glucose and disaccharide maltose. glucose and maltose are useful
food sweeteners and nutrients. Maltose is even usefu:L in culture media.
Glucose is especially useful because it can be isomerized to form the even
sweeter monosaccharide, fructose. this invention relates to a two step
process for obtaining high purity glucose or maltose from a feedstock
comprising starch derived from such plants as cassava, maize, potatoes, rice,
tapioca, taro and wheat.
Figures 1 and 2 are flow diagrams for the conventional process and
the process of this irvention, respeetively.
The conventional scheme for glueose production from thinned stareh
is depieted in Figure 1. In Figure 1, and throughout the specificatioll, the
term "thinned stareh" refers to a liquified stareh partially hydrolyzed by an
alpha-amylase enzyme. Different types of alpha-amylase enzymes are often
used to produce thinned starch with different, but analogous properties
depending upon whether maltose or glucose is desired. lhinned starch has a
dry solids (DS) level of 30-45% and contains a minor proportion of
monosaccharides, up to lOg but usually less than 4%, 20% to 70g disaccharides
through heptasaeeharides (DP2-DP7), and 30% to 80% oeta8aeeharides and the
higher moleeular weight polysaeeharides. One measure of its degree of
hydrolysis is the dextrose equivalent. A thinned stareh is said to have an
inereased dextrose equivalent, that is, an increased proportion of de~tro-
rotatory glueose when eompared to untreated stareh. Untreated starch
contains no or few free units of dextrose; thinned starch may have the
dextrose concentration increased to a low value of from 5 to 25.
In the conventional process of Figure 1, thinned starch 1 enters a
saccharifieation or hydrolysis reactor zone, 2, where it undergoes enzyme-
catalyzed hydrolysis using a glucose-forming enzyme, for example,
.~ I,
amyloglucosidase (glucoamylase), hereafter referred Jo as AG. An
essential feature of present processes is that hydrolysis is continued
in one step until maximum glucose formation is attained, which corresponds
to about 34-96% glucose in the product stream, 3, when using a feed-
stock containing about 30 dry solids. Although only one reactor zone isdepicted or saccharification, this is but one embodiment, and a plurality
of reactor zones in series may be used in other embodiments.
Effluent, 3, from 2 containing more than about 94% glucose (on
a dry solids basis) is then concentrated, where necessary, in an evapora-
tion zone, 4, to afford a product stream, 5, containing from about 35%to about 50% dry solids. This product stream, 5, is typically the feed-
stock entering an isomerization reactor zone, 6, in which glucose is
enzymatically converted to fructose by glucose isomerase.
The conventional preparation of maltose from starch is analogous
to that depicted in Figure 1, the major difference being that the thinned
starch is hydrolyzed with a maltose-producing enzyme. Beta-amylase is
largely or exclusively the maltose-producing enzyme used.
Our invention is directed toward both glucose and maltose produc-
tion. However, solely for the sake of brevity this description following
will be directed toward glucose production, with it being clearly under-
stood that a similar description directed toward maltose production is
contemplated.
Several disadvantages attach to the conventional process. One
disadvantage, generic to any run to maximum conversion, is the increased
cost consequent to the process time requirements for attaining maximum
conversion: the longer the time for such conversion to be established,
the more costly, hence more disadvantageous, is the process. Because
glucose represses enzyme activity by complexing with AG, the hydrolysis
rate is decreased as glucose accumulates and further increases process
time. Another disadvantage characteristic of the relatively lony
residence time associated with the AG-catalyzed hydrolysis of thinned starch,
.3'7~
is that the glucose, a monosaccharide, reverts to disaccharides,
among which is iso~maltose. Because isomaltose is a refractory disac-
charide, that is, it is not readily hydrolyzed, and because it is bitter,
it is a highly undesirable component of a glucose feedstock used for
fructose production. The longer the reaction time, the higher the
glucose level and a higher isomaltose concentration in the product
results.
A still further disadvantage, at least where a soluble enzyme
is used in the saccharification zone, is that the enzyme must be con-
tinuously replaced as it is lost during production. Cost rises directly
as the amount of glucose formed per unit of enzyme decreases.
Because present commercial processes for production of high
fructose corn syrup by isomerization of glucose utilize a glucose feed-
stock containing at least 94% glucose, a constraint of any new or modified
process for production of glucose is that it afford comparable glucose
levels.
The more efficient process of our invention can provide a
product stream haYing at least g4% glucose and avoiding the aforementioned
disadvantages of conventional processes. 0ur invention is a process whereby
a feedstock of thinned starch is hydrolyzed to afford glucose (maltose)
below maximum formation levels, the effluent is separated into a glucose
(maltose)-depleted stream, the glucose (maltose)-enriched stream is
recovered9 and the glucose (maltose)-depleted stream is recycled to
the hydrolysis step.
By carrying out hydrolysis to a state substantially short of
maximum glwcose formation, the invention herein achieves a considerable
saving time and affords glucose with substantially lower levels of rever-
sion products.
~,~?J~ 7~3~.D
Therefore an advantage oF our invention is that it affords
a substantial reduction in process time. Another advantage accompany-
ing a reduction in time is that the process of our invention
affords glucose with less reversion products than the prior art pro-
cesses.
Still other advantages accrue from the characteristics of enzyme-
catalyzed hydrolysis of thinned starch of which presently used commercial
processes cannot take advantage. When a feedstock for AG-catalyzed
hydrolysis is increased in dry solids, it is found that enzyme stability,
as measured by its half-life, also increases. It is also found that the
rate of glucose formation increases with increasing dry solids. Both
of these characteristics are quite favorable, yet cannot be used in
present commercial processes because ;ncreasing dry solids also leads
to a lower maximum glucose level accompanied by increased reversion products.
In contrast to the prior art methods, the process of the instant
invention is able to advantageously utilize the favorable characteristics
of increased enzyme stability and increased glucose formation rate without
any accvmpanying disadvantage of increased reversion products. Thus,
in this sense our invention is truly synerg;st;c; it incorporates the
benefic;al effects without incorporating the detrimental ones.
he characteristic of using immobilized AG in hydrolyzing
thinned starch is that it typically affords less than 94% glucose at
equilibrium and the heretofore relatively long residence time has re-
sulted in the appearance of unwanted reYersion products. Thus, immobili ed
AG can be used only with difficulty in present commercial processes.
Therefore, yet another advantage of the instant invention is that it
readily permits the use of an immobilized AG. One oharacteristic of using
soluble (or unimmobilized) AG in hydrolyzing thinned starch is that ;t is
necessary to continuously replace the enzyme which is lost during the
production of glucose. Therefore, still another advantage of the process
7~
described herein ls that it affords a substantial increase in productivity,
defined as the amount of glucose formed per unit of enzyme. Thls pro-
ductivity increase results, in part, from recycling the enzyme incidental
to the recycle stage of the process (where soluble AG is used), as well
as a longer half-life (where either a soluble or immobilized AG is used).
The glucose level in our process product stream as described
is at least 90%. However, glucose levels of greater than 99% may be
readily achieved by suitably varying process variables. Thus, still
another advantage of our process is that it may be tailored to continually
produce high-purity glucose, with a glucose purity greater than 99%
being attainable.
Yet another advantage of the process which is our invention is
that it can afford virtually complete conversion of starch to glucose.
It should be readily apparent from the multitude of the
aforementioned advantages that our invention represents a substantial
advance in the art of producing glucose at levels of about 94% and
greater by enzyme-catalyzed hydrolysis of thinned starch.
SUMMARY OF THE INVENTION
One embodiment of our invention comprises a process for selectively
obtaining a sugar which is either glucose or maltose from thinned starch.
A feedstock of thinned starch is hydrolyzed under the action of a
glucose- or maltose-producing enzyme to form an effluent stream containing
50% to 92% of the sugar. The effluent stream is separated into a sugar-
enriched product stream and a sugar-depleted stream. The sugar-enriched
product stream is recovered and the sugar-depleted stream is recycled to
the hydrolysis step.
In a more specific embodiment, the effluent from the hydrolysis
step contains 70% to 80% sugar. In a still more spec;fic embodiment, the
sugar-enriched product stream contains at least 90% sugar.
~3~7~6
DESCRIPTION OF THE INVENTIO'I
This invention is a process for obtaining glucose or maltose
from thinned starch which represents a radical departure from prior
art methods. One point of departure is the partial hydrolysis of thinned
starch. That is to say, whereas the prior art methods hydrolyzed
thinned starch for a time sufficient to attain maximum formation of
glucose, the process herein continues hydrolysis or a substantially
lesser period of time, thereby affording an effluent which contains
less than maximum levels of glucose. Yet another point of departure is
the separation of the hydrolysis product into a glucose-enriched product stream
and a glucose-depleted stream with recycling of the latter to the
hydrolysis step. The process herein is conveniently summarized by the
flow diagram depicted in Figure 2.
In Figure 2, a thinned starch, 11, as defined within, usually
containing from about 30% to about 45% dry solids, is the feedstock for
a saccharification reactor zone, 12. Although only one reactor zone
is depicted, this is but one embodiment. Embodiments where a plurality
of reactor zones are used in the saccharification step are contemplated
and are to be considered within the scope of the claimed invention. Ef~lu-
ent stream 13, from the saccharification zone contains glucose at levels
based on total solids from about 50~ to about ~2%, and this is used as
the feedstock for the separation step, 15. Separation is here depicted
as a single stage, but embodiments employing multistage separation are
variants within the scope of this invention.
The effluent from the separation zone is in two streams, one
being product enriched in glucose, 16, to contain at least 90% glucose
(on a total solids basis), the other being a glucose-depleted stream, 14.
This latter is then recycled to the saccharification reactor zone, 12.
~L2~ 3~
Where the glucose-enriched product stream 16 is ultimately
to be used as the Feedstock for a glucose isomerase reactor, it is then
sent to an evaporation zone, 17, where necessary to afford stream 18
containing from about 35% to about 50% dry solids. Said stream 18
then is utilized as the feedstock for an isomerization reactor zone 19
converting glucose to fructose. However, it is to be clearly under- 1
stood that the glucose-enriched product stream may serve as the glucose
source for purposes other than use as a feedskock or isomer;zation to
fructose. Other purposes to which the glucose may be put include hdyrogen-
ation to sorbitol, fermentation to ethanol, and a sweetener in food.
Where the product is maltose there is no isomerization reactor
zone 19 as depicted in Figure 2. The maltose-enriched product stream
corresponding to 16 is either used as is or sent to an evaporation
zone 17 to afford more concentrated solutions of maltose9 or even
dry maltose.
In the initial step of our process, a feedstock of thinned
starch is selectively enzymatically hydrolyzed by a glucose-prbducing
enzyme, chiefly AG, to an effluent containing from about 50% to about
92% glucose. This selective hydrolysis step often is referred to as a
saccharification step. The AG used nay be soluble, in which case it is
recycled with the glucose-depleted stream, or it may be an immobilized AG.
In either case pullulanase or alpha-amylase, or both, may be present in
the thinned starch feedstock to aid hydrolysis. The temperature at which
the enzymatic hydrolysis is conducted depends upon the thermal stability
of the enzyme used, but generally the temperature is between about 40
and 80C, with the temperature of about 60C being the most usual one.
however, the AG from at least one microorganism is known to be sufficiently
thermostable to allow the process to be run at temperatures even up
to about 100C. The pressure at which enzymatic hydrolysis is conducted is
from 1 to 1000 pounds per square inch. the residence time during which the
--7--
23~7~-3~
starch is in contact with lmmobilized enzyme is relatively short, that is,
in a range of from 4 minutes to 1.6 hours and is in correlation with liguid
hourly space velocities which may range from about 2 to about S0.
The space velocity and residence time are correlated in such a
manner, i.e., short resiclence time and high space velocity within the ranges
hereinbefore set forth so as to provide a conversion rate within the desired
range. Conversely, it is also contemplated that long residence times and low
space velocities with the aforesaid range may also be employed to effect
the desired result.
The residence time during which the starch is in contact with soluble
enzyme is relatively short in comparison to conYentional processes, that is,
in a range of from 2 hours to 48 hours.
A desirable consequence of hydrolyzing thinned starch to an
effluent containing from 50% to 92% glucose is a substan-
tial reduction in reaction $ime. Thus, hydrolysis to a product con-
taining 50% glucose may take less than one-fourth of the time needed
to attain 94% glucose, hydrolysis to 90~ glucose may take only one-
half the time, and even hydrolysis to 92% glucose may take only three-
fifths the time. Another desirable consequence is a decided improve-
ment in organoleptic characteristics through substantial reduction of
the bitter principal, isomaltose9 hydrolysis to 50~ glucose may be
accompanied by only about one-fourth as much isomaltose as accompanies
the 94% glucose product.
A feedstock of thinned starch containing from about 30% to
about 45% dry solids is conventional in the industry, although our pro-
cess is not limited thereto. However, a dry solids level from about35% to about 45% is preferred in the practice of this invention to
obtain the full advantage of the salutary effect of higher dry solids
on enzyme stability and the rate of glucose formation.
Prior art methods continue the hydrolysis to maxlmum flu-
cose formation, which corresponds to glucose leYels of about 94%. How-
ever, an essential feature of our process is continuation of hydrolysis
to an effluent containing from 50% to 92%, but usually not more
than 90% glucose. An effluent containing from about 60% to about 85%
glucose is deisrable, and one containing from aout 70% to about 80
glucose is particularly preferred.
The hydrolysis effluent recovered from this saccharification
step is then separated into a glucose-enriched product stream and a
glucose-depleted stream in a separation step. Where the glucose-
enriched product stream is used as the feedstock for isomerization to
fructose, the stream will c.ontain at least about 94% glucose since
present processes for forming fructose from glucose require a feedstock
containing a least about that level of glucose purity.
Where a feedstock of less Han 94% glucose is acceptable,
a less stringent separation to afford 10wer purity glucose may be
effected. As a practical matter9 the glucose-enriched product stream
generally will contain at least about 90X glucose. It also must be
understood that separation may be performed to obtain higher purity
glucose, i.e., 94~ glucose. In fact, our invention may be used to
continually produce glucose of greater than 99% purity where such
high purity material is desired.
Generally, this separation step will comprise a single stage.
Hohever, multi-stage separation may be advantageous in some circumstances,
and these are considered to be within the scope of our invention. In any
event, the glucose-enriched product stream from the separation step
is recovered for subsequent use or processing.
Any method of separation which is selectiYe for glucose rel-
ative to disaccharides and higher polysaccharides is suitable. where
maltose, a disaccharide. is formed the separation needs to be selec-
tive relatiYe only to higher polysaccharides. For example, a membrane-
based separation may be effectively utili2ed. As another example, a
separation based on solid adsorbents may be utilized. Examples of
_g_
3 7
the latter include aluminas, silicas, various clays, zeolites, and so
forth. Still another method of separation is selective crystalliza-
tion of glucose prom the saccharide mixture. Still other methods r
of separation which may be used in the process herein include solvent
extraction and supercritical extraction, to cite but two further
exemplary methods.
An integral part of this invention is the recycling of the
glucose-depleted stream to the hydrolysis step. When a plurality
of hydrolysis zones connected in series flow are used in the hydrolysis
or saccharification step, the particular hydrolysis zone which is the
entry point for the recycled stream will depend on process parameters
such as reactor configuration, the activity of the particular enzyme,
the concentration of reactants and/or products in the recycle stream,
whether the enzyme is soluble or immobilized, the enzyme concentration
if soluble, desired purity of the product, the particular means of
separation, and so forth. Under many process conditions the location
of the recycle point may be varied broadly without substantial impact.
or instance, where the enzyme i5 immobilized, a portion of the glucose-
depleted stream may be recycled to join the feed prior to entering the
hydrolysis zone. But in any eYent it should be clear that the determina-
tion of a suitable entry point will depend upon the specific parameters
utilized in any particular process with its determinat;on well within
the capability of the skilled worker.
As heretofore set forth, the enzymes which are utilized in
the process of this invention most often comprise amyloglucosidase or
betaamylase which may be composited on a solid support. By utilizing
enzymes which are immobilized on a support, it is possible to stabilize
the enzyme in a relative manner and therefore to permit effective use
of enzyme which otherwise might be lost in the reaction medium. Such
immobilized or insolubilized enzymes may be employed in various reactor
--1 0--
3t~ Ç~
systems such as in packed columns, stirrinq tank reactors, etc., depend--
ing upon the nature of the substrhte which is utilized therein. By per
mitting the reuse of the enzymes which are in a relatively stable condi-
tion and thus may be utilized for a relatively lengthy period of time,
it is possible to operate the process in a commercially attractive
and economical manner.
The particular enzyme may be immobilized on a solid support
in any manner known in the art. One such method of immobilizing the
enzyme which may be used as an illustration of a method for immobilizing
the enzyme is taught in U.S. Patent 4,141,~57.
As hereinbefore set forth, it is also contemplated withir, the
scope of this invention that ultra-filtration membranes may be used
to recover glucose or maltose from a partially hydrolyzed
reaction mixture resulting from the treatment of a liquid
starch feedstock with an enzyme. Any membrane which possesses
an appropriate molecular weight as well as pore sizes capable of yielding
the desired high glucose or maltose content as the permeate while retaining
the rejected material which contains unhydrolyzed oligosaccharides is
suitable. The ultra-filtration membrane will possess a molecular weight
cut-off in the range of from about 100 to about 5000 and will also possess
pore sizes in the range of from about 5 to about 125 Angstroms. Some speci-
fic examples of these membranes which may be utilized will include
cellulose acetates such as cellulose diacetate, cellulose triacetate
or mixtures thereof, a membrane resulting from polyethyleneimine cross-
linked with a dialdehyde, a diacidchloride, or a diisocyanate9 polyacrolein,
chitosan cross-linked with a dialdehyde such as glutaraldehyde9 polystyrene-
sulfonates, etc. It is to be understood that these ultra-filtration
membranes are only representative of the class of membranes which may be
employed and that the present invention is not necessarily limited thereto.
--1 1--
The two-step process o the present invention may be effected
in any sultable manner and may comprise either a batch or contlnuous type
operation. For example, when a batch type operation it cmployed, a qunntlty
of the feedstock comprisiug a liquid starch which ha been previously treated
with alpha-amylase to increase the dextrose equivalent is contacted wlth
an lmmobillzed amyloglucoslda~e9 in the event that a syrup high ln glucose
content ls desired, for a predetermined period of time while employing
reaction conditions which include a temperature of from about 45 to about
70C and a pressure which may range from about 1 to about 1000 psl. In
addition, the resldence time during which the feed6tock ~B in contact with
the immobilized enzyme iB correlated wlth the liquid hourly space velocity
at which the feed is introduced Jo as to produce a conversion of the liquid
starch to glucose within the desired range. After passage over the immobllized
enzyme, the effluent iB recovered and the partially hydrolyzed starch is
then subjected to an ultrafiltration step. In thls step, the reactlon
mixture is psssad through sn ultrafiltration membrane o the type hereinbefore
set forth whereby the permeate which possesses a high glucose or maltose
content is recovered as the permeate upon separation from the retentate.
The latter may then also be recycled, a portion of the recycle stream belng
ndmixed with the ef1uent from the enzyme trea~me~t while another poFtion
of the retentate iB admixed with the ieed stream to the immobllized enzyme
treatment zone. It is also contemplated that a third portion, if BO desired,
may be recycled back to the zone ln which the feedstock i8 pretreated wlth
alpha-amylase.
It is also contemplated within the scope of thih in~entlon
that the process may be effected in a continuous manner of operation.
When this type of operation is employed, the feedstock compri~lng the treated
liquified starch i8 continuously charged to a reaction vessel such as a
column which contains the desired immobilized enzyme, said column being
maintained at the proper operating condi-
,~:
- - 12 -
rm~
tions of temperature and pressure. After passage over the enzyme
at a predetermined liquid hourly space velnclty which ls sufficiently
high so as to only partially hydrolyze the feedstock, with a con-
comitant low or nonexistent production of reversion products such
as isomaltose, the effluent is continuously withdrawn and charged
to an ultrafiltration apparatus which contains a membrane of the type
hereinbefore set forth. After passage through this apparatus, which
is also maintained at the proper operating cond;t;ons of temperature
and pressure, the pen~eate comprising syrup which has a high glu-
cose or maltose content, l.e., above 90X, is recovered. The reten-
tate material which contains unhdydrolyzed oligosaccharides such as
those which have a DP rating of DP7, DP8, DPg+ (the designation DP
being the degree of polymer kation) is also recovered and a portion
thereof recycled back to the column containing the immobilized enzyme
for further use as a portion of the feedstock, another portion admixed
with the effluent from the immobilized enzyme treatment and, if so
desired, a portion to the alpha-amylase step for pretreatment.
Each of the heretofore mentioned membrane separations may be
performed using solid adsorbents as well. One such adsorbent is dis-
c10sed in U.K. Patent No. 1,585,369,
and comprises X or Y zeolites containing
one or more selected cations at the exchangeable cationic sites.
Potassium -X zeol;te 7S a particularly preferred adsorbent exhibiting
selectivity for glucose with respect to disaccharides and higher
ol;gosaccharides and the polysaccharides.
The following eleven examples are given for the purpose of thus
trating the present inYention. However, it ;s to be understood that
these examples are given merely for purposes of illustration and that
the present process us not necessarily limited thereto.
I, ,
~L~J~7~3
EXAMPLE I
To illustrate the process of the present invention, a starch
feedstock was treated with alpha-amylase to adjust the dextrose
equivalent to 15 DE. In this case, the enzyme was immobilized and
comprised amylglucosidase composited on a solid alumina support. The
treated starch feedstock was passed through a column of
40 cc of the immobilized enzyme, said starch feedstock con-
taining 0.1% benzoate and 50 ppm of sodium omadine at a pH of 4.2.
The starch was treated at a temperature of 45C, and a pressure
greater than atmospheric at a liquid hourly space velocity of 3.21.
The effluent from this column was analyzed by means of liquid chroma-
tography. The analysis showed the following area in which DP is
the degree of polymerization (or examp1e, DP7 = seven ~7) monomers
of glucose in the oligosaccharide):
9+ 8 7 DP6 DP~ DP4 DP3 DP2 Glucose
19.1 0.2 0.1 - - - 0.3 3.1 77.2
l The e~luent in an amount ox 200 cc which was recovered from
this column was then passed through a cellulose acetate membrane,
1~0.7 cc out ox the original 200 cc being obtained and constituting
the permeate. Analysis of the permeate aFter passage through the mem-
brane at a temperature of 22C and a pressure of 9~ psi showed that
said penmeate contained 94.2% glucose, 3.6% maltDse and only 1.7% of
the oligosaccharides having a DP of 9~.
The retentate which is recovered from the treatment with
the cellulose acetate membranes may then be recycled and utilized as
a portion of the feedstock which is charged ts the zone containing
the immobilized enzyme comprising amyloglucosidase composited on the
treated alumina support.
-14-
7~
EXAMPLE II
In a manner similar to that hereinbefore set forth in Ex-
ample I, a treated liqui~ied starch ~eedstock was again passed
through an lmmobilized amyloglucosidase column under conditions sim-
ilar to those in Example I. The effluent from this column, which
contained 77.2% glucose as well as minor amounts of maltose, DP3,
DP7, DP8 and a major amount of DPg+oligosaccharides, was passed
through a membrane comprising polystyrenesulfonate and sold under
the trade mark Amicon UM2. The volume ox the effluent which passed
through the membrane was 122.8 cc. Analysis of the permeate by means
of liquid chromatography showed 97.6~ glucose, 2.3 maltose, and only
0.1% of DPg+ oligosaccharides.
When the above experiment was repeated using a membrane
comprising cellulose acetate sold under the trade name Nuclepore and
the effluent from the immobilized amyloglucosidase column was passed
over this membrane at a temperature of 22C and a pressure of 90 psi,
the penmeate was found to contain 97.5% glucose, 2.3% maltose, and
0.2% DP9~ oligosaccharides.
Likewise, the retentate which may be recovered from the
treatment of the effluent with the cellulose acetate membrane may be
recovered and recycled a portion of the retentate being admixed with
the effluent from the immobilized enzyme treatment while the other
portion is recycled to form a portion of the ~eedstock which is
charged to the inmobilized enzyme treatment zone.
EXAMPLE I_
TD illustrate the ability Do the process of the present in-
-15-
~L~3~7q~
vention to recover a product containing a high maltose concentration,
a liquified starch feedstock which had been pretreated with alpha-
amylase to adjust the dextrose equivalent to the desired level was
passed through a column containing 100 cc of beta-amylase immobilized
on a solid matrix similar to that set forth in Example I above. The
feedstock which had a pH of 5.0 contained 0.1 mole ox acetate and 0.2
mole of benzoate which acted as a buffer. The feedstock was passed
over the enzyme at a temperature of 55C, a pressure greater than atmo-
spheric, at a liquid hcurly space velocity of 5. The effluent which
was recovered from this column was subjected to liquid chromatography
analysis with the following result:
HPLC ANALYSIS: (AREA %)
9~ B 7 DP6 DP5 DP4 DP3 DP2 Glucose
36.9 0.9 0.3 , 0.3 0.1 0.5 11.1 50.2 O5
The effluent from this column in an amount of 100 cc was
passed through a cellulose acetate membrane while maintaining the mem-
brane apparatus at a temperature of 22C and a pressure of gO psi.
Analysis of the permeate by means of liquid chromatography showed the
presence of 79.0% maltosep 16.3% of a DP3 product and 2.4% of a DPg+
oligosaccharide.
When the aboYe experiment was repeated using an ultraf;l-
tration membrane sold under the trade mark of Amicon UM2, the permeate
was found to contain 90.1% maltosep 9.7~ of a DP3 oligosaccharide and
only 0.2X of a DP9~ oligosaccharide.
In a similar manner the retentate which is recovered from
the treatment with the ultrafiltration membrane of the above paragraph
-16-
may be recycled, a port;on being admixed with the effluent withdrawn
from the treatment with the immobilized beta-amylase while another
portion may be admixed with the feedstock comprising the liquified
starch prior to passage over the immobilized enzyme.
EXAMPLE IV
In this example, a liquified starch feedstock which may be
pretreated with alpha-amylase Jo adjust the dextrose equivalent of said
starch to a predetermined level may be passed through an immobilized
amyloglucosidase column at a temperature of 45C and a pressure greater
than atmospheric. The effluent from the column may be withdrawn after
having been in contact with the immobilized enzyme for a predetermined
period of time sufficient to permit a conversion to glucose of about
75% and may then be passed through an ultrafiltration membrane compris-
ing polyacrolein, said passage over the membrane being effected at con-
ditions which Jill include ambient temperature and a pressure of about
2~ psi. The permeate may be recovered while the retentate may ye re-
cycled to be admixed with the effluent prior to passage over the afore-
sa;d membrane.
In a similar manner, a liquified starch feedstock which con-
tains a dextrose equivalent of about 15 may also be passed over an im-
mobilized enzyme comprising beta-amylase composited on a treated alu-
mina support, the reaction conditions including a temperature of about
50C and a pressure greater than atmospherk. After passage over the
enzyme it a predetermined liquid hourly space velocity and for a resi-
dence time sufficient to permit a conversion of about 75% of the
starch to glucose, the effluent may be continuously withdrawn and pas-
sed over the membrane comprising polyethyleneimine dialdehyde, said
-17-
3~7~ 3~
passage over the membrane being effected at a temperature of about
25C and a pressure of about 100 psi. The permeate which may contain
over 90% glucose may be recovered while the retentate may be recycled,
one portion of which may be admixed with the effluent, withdrawn from
the enzyme treatment zone, a second portion may be recycled to be ad-
mixed with the liquified starch feedstock entering the immobilized en-
zyme zone, while a third portion may be recycled to the pretreatment
20ne in which the starch feedstock is contacted with alpha-amylase,
the alpha-amylase acting to raisP the dextrose equivalent of the starch
to a predetermined level.
The effluent which is continuously withdrawn from the treat-
ment with the immobilized enzyme may also be passed over an ultrafil-
tration membrane comprising chitosan dialdehyde at reaction conditions
which include a temperature of bout 22C and a pressure of about 100
psi, the permeate oomprising a major portion of glucose being re-
covered while the retentate may be recycled to be admixed with the ef-
fluent which is dispersed from the i~obilized enzyme treatment 70ne.
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l 3~ J
The seven examples whlch follow are merely illustrative of this
invention and are not intended to limit or rest;rict it in any way. ,~
Thinned starch used as the feedstock was Maltrin-150, a
typical analysis for which showed about 1% glucose, 3% DP2, 6% DP3,
4Y~ DP4, 4% DP5, 9% DP6, 16% DP7, and 58D/o DP~ and hiyher.
Glucoamylase was assayed as follows. Jo 4 ml of a starch
solution, 300fD dry solids, was added 25 microliters of the enzymP solu-
tion, and the mixture was incubated 30 minutes at 6~C. Hydrolysis
was quenched bythe addition of 1 ml 0.2N NaOH, and the mixture was
cooled. The amount of glucose formed was determined using a glucose
analyzer. The number of grams of glucose produced per hour is the AG
activity expressed in Miles units.
EXAMPLE V
-
A solution of AG (77 units/liter) at pH 4.2 in glucose so-
lutions containing varying levels of dry solids was maintained at 60~C.
Aliquots were withdrawn periodically and assayed for AG activity. the
enzyme half-life was found to be 4.8, 12.3, and 21.9 days at dry solids
levels of 30%, 44% and 55%, respectively. Thus, enzyme stab;lity as
measured by halt e at 60~G is increased over 4.5-fold in going from
30D/. to 55% dry solids.
* trade mark
_19_
g~3q~
EXAMPLE Vl
Feedstocks of Maltri~-150*of different dry solids (DS) level
at pH 4.2 containing AG at 77 units per liter were hydrolyzed at 60C.
glucose concentration as determined by high pressure liquid chromatog-
raphy was monitnred with time, the results being su~arized in the ac-
5companying table.
TABLE 1. Glucose Concentration With Hydrolysis Time
Time Glucose conc., grams/liter
(hours) 30% DS 44% DS 55% DS
26~ ~90 3~5
320 405 42~
330 ~40
340 525 600
340 525 ~00
The results clearly show that glucose production rates in-
creases with increasing dry solids level of fe dstock.
EXAMPLE Vll
A feedstock of Maltrin-150* 30% dry solids9 at pH 4.2 and
containing AG at 77 units per ljter was hydrolyzed at 60C. Product
was analyzed for glucose, total disaccharides 5DP?), and isomaltose.
Results using two different lots of AG are summarized in the table
below.
* trade mark
-20-
l 3~;:3~3 1
TABLE 2. Glucose, Disaccharide, and Isomaltose Production
_ _ _ _
Time Weight %
Run 1 (hours) ,glucosed;saccharides isornaltose
2 28 ~.5 0
58 9.0 0.6
6 7~ 4.5 0.9
88 4.0 0.9
lQ 90 4.5 1.3
12 92 4.R 1.3
5.8 ~.2
9~ 7.0 3.
Run 2 1.8 45.1 17.3 0
4.7 69.0 10.8 0
5.6 74.~ 7.5 0
7.5 8~.9 ~.5
13.0 91.1 3.2 ~.0
20.0 93.5 3.4 O.B
- ~8.0 94.8 4.1 1.6
Thus, relative to the time needed to attain a 94% glucose
product attainment of a 90% glucose product takes bout half the time,
and attainment of a 92% glucose product takes about 60% Df the time.
These results also clearly show the accumulation of is~mal-
tose during the latter stages of conversion.
-21-
~.~3 7~ D
EXANPLE VIII
lhis exnmyle compares results from a once-through reactor,
to a conventional operatlon in which thinned starch is hydrolyzed to
about 94% or greater glucose, with a clo~ed-loop system where reactor eEfllJent
iB Kent to a membrane, a gluco~e-enrlched product strenm iB drawn off,
and the gluco~e-depleted stream is recycled to the beginnlng of the reactor.
All hydrolyses were performed at 60C, pH 4.5~5.5. Run A 1B the conven-
tional, once-through reactor; both Runs B and C are closed-loop ~y~ems,
wlth B using a cellulo6e acetate membrane hazing a 10,000 molecular weight
cutoff operating at 60C, 150 prig, and C UBing a polyelec~rolyte membrane
with a 500 molecular weight cutoff operatlng at 60C, 300 psig, both supplied
by Amicon Co. under their trade marks YM10 and UN05, respectively Results
are listed in Table 3, where DPl repre~entR total mono~accharldes, nearly
all of which is gluco6e7 DP~ represents disaccharides, DP~ are poly-
saccharides of four or more units, end DS i8 dry solits. For Runs B and
C reactor effluent, product and recycle stream analyses are equilibrium
values .
- 22 -
rm/Ad
TABLE 3. Companion of Once-Through (Single Pays)
Reactor With Clo~ed-Loop, Recycle Reactor
Run A Run B Run C
enzyme (AG) dosage, unlt~/llter 77 34 58
Residence time, hours 48 14 20
Feedstock Composition (%)
DS 30.0 26.1 24.7
DPl 1.0 1.2 1.6
DP2 4-5 4~3 3-9
DP4+ 87.5 87.8 88.3
Reactor Effluent
DS 33.0 27.0 30.4
DP 95.7 90.5 83.4
DPl 4.1 2.9 4.4
DP4+ 0.2 6.3 11.6
Product Stream
DS 26.1 24.7
DP 93.6 98.0
DPl 3.1 2.0
DP4+ 2.~ 0.0
Recycle Stream
DS 27.4 34.2
DPl 89.9 82.5
DP2 3.5 3.8
DP4+ 6.3 13.1
EXAMPLE IX
A kinetic model was developed from experime~$al data which
not only closely reproduced exiting experimental dnta but alto had reliably
hlgh predictive abillty. Among the variable (experlmental parameters)
accommodated by the model were feedstock (thinned starch) composltlon,
enzyme dotage, reactor re~ldence tlme, membrane type and operating condition.
- 23 -
` .j.
. .
rm/~
Output included glucose and 1somaltose concentration in the p~odl1ct strenm.
The feedstock of thinned starch hod 30~6 weight percent dry solids with
1.2% DPl, 4.8X DP2, and 87~ DP~. Run A represents a once through reactor,
nnd Runs B and C represent a closed-loop recyc]e reactor usi11g the polyelectrolyte
membrane described in tke prior example with the glucose-depleted streRm
recycled to the top of the reactor. Reactor efflue~ and product compositions
are equillbrlum values.
TABLE 4. Isomaltose Accumulation
Run A Run B Run C
AG concentration, units/liter 77 9 18
Residence time, hours 48 lO lO
Reactor effluent: % glucose 95.774 87.3
% isomalto6e 1.66 0.7 l.B5
Product % glucose 95.5 97.2
% isomaltose 0.34 0~73
These data clearly show that the closed-loop, recycle process
produces glucose at as high concentration as that from a conventional,
once-through reactor at substantially lower enzyme dotage, with one-fourth
the isomaltose content. In fact, Run C 8hows that one can lncrease glucose
in .he product to over 97% (using one-fourtb the enzyme concentration)
and still have less than half the isomaltose content a that from the con-
ventional process.
EXAMPLE X
In this example the aforementioned kinetic model was used to
determlne the effect of recycle location on glucose and isomaltose concen-
tration ln the Rteady state product and reactor effluent streams. Data
- 24 -
rm~ia
~ri~t7
re summarized ln Table 5, where "reactor volume" refers to the percent
reactor volume prior to the recycle loc~tlo~; 0 represellts the top, 100
represents the bottom of the reactor. Thinnad starch feed~tock had the
composition of that in the foregolng example; reactor time was lO hour
and the polyelectrolyte membrane of example 4 way used under the stated
condition
TABLE 5. Effect of Recycle Point on Product Composltion
0 30 50 70 ~0 95
AG concentratlon: 18 u/l
Reactor effluent
glucose 87.4 88.0 87.0 85.0 73.0 57.0
isomaltose 1.85 1.8 1.7 1.3 0.6 ~25
Product
glucose 97.2 97.2 97.2 96.9 96.9 90.7
isomaltoee 0.73 0~71 0.66 0.55 0.27 0.13
AG concentratlon: 77 u/l
Reactor effluent
glucose 91.5 91.5 91.5 91.5 92.3 91.0
isomaltose 4.75 4.8 4.7 4.4 3.35 2.5
Product
glucose 96.9 96.9 9o.9 97.0 97.3 97.4
isomaltose 1.73 1.74 1.70 1.59 1.22 0.93
The results it the table reflece the iact thae at high AG con-
centration the product eomposition i8 rather insensitive to recycle point
throughout, and at lower enzyme concentration no substantial change in
product composition is seen when the recycle point is between 0-90% of
reactor volume. The overall conclusion iR that recycle location will be
a process choice depending upon other process parameter such as enzyme
concentration, residence time, separation means used, product composition
desired, etc.
- 25 -
f 3 16
EXAMPLE XI
These data generated by the kinetic model show the advan-
tages,absent in a once-through reactor,accruing from a feedstock with
high dry solids used in a closed loop, recycle process. F~eds~ock
had the composition of Example 5 with only the dry solids (weight
percent) varying. the recycle reactor utilixed the polyelectr~lyte
membrane nf Example 4 under the conditions stated therein.
TABLE 6. Effect of Dry Solids Variation
Closed lop
Once-~hrough reactor recycle reactor
% Dry solids 30 40 40
Residence time, hours 48 71 10
AG concentratinn, units/liter 77 77 18
Product, wt. I: glucose 95.7 93.6 95.6
isomaltose 1.7 .2.2 0.7
In a ~nce-through reactor residence time is unacceptably long
end isamaltose toncentration is unacceptably high using 40~ dry solids
fe~dstock~` whereas in the closed loop recycle reactor configuration
residence time is reduced told, isomaltose concentration 3-~old. and
enzyme concentration 4-~old.
-~6-
