Note: Descriptions are shown in the official language in which they were submitted.
J-3332
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CARBOHYDRATE PROCESS
This invention is concerned with a new and useful
process for the production of glucosone and more particularly
for the production of glucosone from which food-gxade fructose
5 can be obtained.
Commercial methods for the produetion of fruetose,
a commercially important sweetner, primarily involve a two-step
process, the first, hydrolysis of a polysaccharide such as
starch to produce glucose and the second, isomerization of the
10 so-produced glucose to form fructose. The latter step, as
is well-known, produces a mixture of glucose and ~ructose
from whieh it is difficult to separate the desired product,
fruclose. The commereial separation rethod involves the use
of crystallization techniques which are costly and time-consuming.
15 More detailed description of the various methods of isomerizing
glucose can be found in the literature, e.g., U.S. Patent
3,788,3459 and 3,616,221.
Glucose can also b~ converted to fructose by the
aetion of an enzyme, designated glueose-2-oxidase, to form
20 glueosone (D-arabino-2-hexosulose) whieh in turn ean be re-
dueed to fruetose with zine and aeetic aeid [Folia Mierobiol.
23, 292-298 (1978) and Czechoslovakian Patent No. 175897 to
- Volc et al.].
The reaction of glueose-2-oxidase with glueose to
25 produee glucosone also yields hydrogen peroxide in equimolar
amount. The use of the so-produeed hydrogen peroxide in the
eonversion of alkenes to eorresponding halohydrins and epoxides
has been proposed in European Patent Applieation 7176. In
the published application, the in situ formation of hydrogen
3 peroxide is proposed by inclusion of glucose-2-oxidase and
glucn~e in the reaction mixture whieh ineludes a halogenating
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1 enzyme and a source of inorganic halide into which the selected
alkene is to be introduced. The disclosure of the European
patent applic~tl~n fnrther ;ndiG~te~ that the gl-lcosone pro-
duct of the enzymatic oxidation of glucose can be converted
5 to fructose by simple chemical hydrogenation.
However, fructose produced by the said process can
be contaminated with significant amounts of by-products from
both the enzymatic conversion of glucose and the alkene con-
version reaction. In particular, the latter reaction produces
10 halohydrins and alkylene oxides, e.g. ethylene oxide, which
are highly toxic materials even at levels in the region of
parts per million. Thus, fructose produced by such a process
will require careful and costly purification to attain food-
grade purity. Further, the potential for contamination of
15 fructose by virtue of secondary reactions during the initial
processing stage is quite high due to the highly reactive
products, halohydrins and alkyleneoxides, and substantial
purification procedures are required to assure the high level
purity required for food grade fructose.
20 S~MMARY OF THE INVENTION
This invention provides a method for the production
of glucosone by enzymatic oxidation of glucose to glucosone in
a reaction zone from which hydrogen peroxide is removed by
use of a hydrogen peroxide-permeable membrane into a second
25 reac~ion zone.
In accordance with one embodiment of the invention,
the second reaction zone contains a reducing agent ~or the
hydrogen peroxide which migrates through the semi-permeable
membrane. The presence of the reducing agent in the second
30 zone encourages a faster migration of the hydrogen peroxide
out of the first zone. Reducing agents for this embodiment
' are well-known to those skilled in the art and include a
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1 variety of systems such as organic reducing agents, anions,
cations and enzymes. Organic reducing agents are exemplified
by aldenyaes which are readily oxidized to corresponding car-
boxylic acids. Reducing anions include, for example, oxalate,
5 sulfite, phosphite and iodide ions. Reducing cations include
a wide variety of cations which can exist in variable valence
states such as the transition metals Fe, Co, Ni, Cr and the like.
Re~ucins enzymes are readily available from a variety of natural
products and include catalase and peroxidase. Catalase is
10 found in yeast, eggs and blood while peroxidase is found in
horseradish.
The quantity of reducing agents in the second zone is
not critical but it is preferably used at levels to significantly
reduce the amount of hydrogen peroxide produced in the first
15reaction zone. Thus, stoichiometric quantities of reducing
agent will assure more complete removal of hydrogen perbxide
from the first reaction zone and the use of even excess amounts
over the stoichiometric will be practical particularly in those
cases where the selected reducing agent is readily available
20and economical.
Of course, the use of less than stoichiometric
quantities of the reducing agent is also within-the scope of
the invention but will be less efficient.
The membranes employed in the present process are
25for the purpose of establishing two separate zones and per-
mitting migration of hydrogen peroxide from the first to the
second zone. The membranes therefore should be of suitable pore
size to selectively permit hydrogen peroxide migration, but
to preclude passage of larger molecules in the first reaction
3zone. Such membranes are readily available oo~mercially and
can be defined in terms of the molecul~r welght of sol~te
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1 particles to pass through the membrane. In the present inven-
tion, membranes which permit substances of a molecular weight
of less than about 100 are to be used, and preferably less than 50.
The migration or passage of hydrogen peroxide through
5 the aforesaid me~brane is accomplished through establishment
of an equilibrium predicated on the relative concentrations
of H202 on each side of the membrane. As the concentration
of hydrogen peroxide in the first zone increases, the H202
tends to migrate to the second zone until equilibrium is rees-
10 tablished. The inclusion of a reducing agent in the secondzone increases the rate of flow of hydrogen peroxide through
the membrane by offsetting the equilibrium in the direction of
the second zone, for which reason the reducing agent embodiment
is generally preferred.
Employing the present process results in considerable
advantage particularly in the further processing of glucosone
to frucLose. The migration of hydrogen peroxide from the first
reaction zone of course affects the rate of the enzymatic oxi-
dation of glucose so that the reaction tends to be more complete
20 and the reaction times can be shorter than normally required.
Further, the first reaction zone is essentially free of con-
taminants that will accumulate primarily in the second reaction
zone where the so produced hydrogen peroxide is reacted. The
glucosone solution produced in the first reaction zone can be
25 used as such in the hydrogenation step or can be concentrated
or otherwise processed as desired. The glucosone solution is
substantially free of contaminants other than some unreacted
glucose, or glucose dimer or trimer, and whatever contaminants
that may have been introduced in the original glucose charge.
30 Usually, the glucose charge will be a hydrolysate of a natural
product containing glucose units, most commonly starch, which
~ will contain soluble contaminants such as other carbohydrates,
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le.g. maltose, formed in the starch hydro]ysis.
Accordingly, the rèduction of the reaction product
of the fir~t ~ne will provi~e a product, frllctnse which ~il1 he
comparatively free of contaminants that effect food grade
5status for the product, the contaminants being derived
only from the glucose natural sources, e.g. starches such
as corn starch.
PREFERRED EMsoDI~IENTS
The membranes to be used in the ~resent process
10are any of those commonly employed in aqueous systems and
include a wide variety. Most commonly, the membranes will be
comprised of nylon, a styrene polymer, usually polystyrene
teflon, or a cellulose ester such as cellulose acetate or
propionate. In a first embodiment, the membrane is fitted
15into a reactor to provide two zones in a manner to preclude
unintended mixing of the contents of the two zones. In a
second embodiment, separate reactors can be coupled with the
selected membrane providing the requisite interface in the
coupling. For maximum migration of hydrogen peroxide from
20 the first zone to the second zone, membranes of significant
exposed surface area are of course preferred for which reason
the first embodiment is more preferable.
The glucose-2-oxidase enzyme can be provided in ~he
form of the enzyme solution in water, immobilized enzyme or
25immobilized cells or mycelium or the free cells or mycelium.
Most commonly since the enzyme is intracellular, the cells or
mycelium of the selected microorganism are used by merely
suspending them in the reaction solution. Promoters and pro-
tectors for the enzyme can also be present. For example, as
3 described in the aforesaid Folia Microbiol. 23, 292-298 (1978),
the presence of fluoride ion promotes the enzymatic oxidation
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1 of glucose with _. mucida. Protectors for enzymes can also be
used, e.g. Co, Mn and Mg salts.
- The enzym~tic o~. dation ïeaC~iull .is ~arried out until
substantially complete as can be determined by monitoring the
5 mixture using aliquots to test for glucose content~ or by
colorimetric determination of glucosone or by determination of
of hydrogen ~erûxide. Usually, reaction periods of about 24-
48 hours are sufficent, depending on enzyme potency or activity.
A wide variety of microorganisms can be used to pro-
lO duce the glucose-2-oxidase employed in the present process.
For example, the following organisms are described in the
literature for this purpose:
I Aspergillus parasiticus [Biochem. J. 31, 1033 (1937)]
II Iridophycus flaccidum rScience 124, 171 (1956)]
15 III Oudemansiella mucida [Folia Microbiol. 13, 334 (1968)
ibid. 23, 292-298 (1978)]
IV Gluconobacter roseus [J. Gen Appl. Microbiol. 1,152 (1955)]
V Polyporus obtusus [Biochem. Biophys. Acta 167, 501 (1968)]
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VI Corticium caeruleum ~Phytochemistry 1977 Vol. 16, p 1895-7]
The temperature for the enzymatic oxidation reaction
is not critical. The reaction can be conducted at room tempera-
ture, or even somewhat higher than room temperature where the
enzyme system employed is of reasonable heat stability. In
2sparticular, it is preferable to operate at 50C. ~nd above.
with heat stable enzyme systems in which range bacterial infection
of the reaction mixture is minimized. Alternatively, the enzy-
matic reaction mixture can contain antibacterial agents to pre-
clude extensive bacterial growth.
3o The first reaction zone of course should contain no
significant amounts of a reducing agent for hydrogen peroxide
~ so that the beneficial results of the present process can be
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1 realised. Thus, the system should be substantially free of
reducing agents for H2O2, i.e. a non reducing system.
During the course of the present process, it is
possible for some diffusion of material from the second reaction
5 zone into the first zone, especially where anions, cations or
low molecular weight reducing agents are present in the second
zone. Therefore, it is usually preferred to use reducing agents
wllich either do not diffuse, e.g. peroxidase and catalase enzymes,
particularly in the immobilized form, or which on oxidation form
lO food-acceptable products, e.g. ferric or sulfate which derive
from ferrous and sulfite ions on oxidation by H2O2. By this
preferred procedure, undesirable contamination of the first
reaction zone is reasonably avoided.
In a particular preferred form of the invention the se~a-
rated hydrogen peroxide is reacted with yeast,whole milk or eggs
to effect pasteurization at room temperature with the obvious
advantage of avoiding elevated temperature normally used for
pasteurization. Thus, the yeast, milk and the eg~s eacl~ ar2
capable of acting as the reducing agent in the second zone.
The reduction of glucosone to fructose is accomplished
by known procedures including chemical reduction as with zinc
and acetic acid as well as catalytic hydrogenation, with the
usual metal catalysts. Of these, the preferred metal catalyst
is Raney Ni since its use is compatible with the desired food
2~grade of fructose, i.e. no residues or contaminants are left
by this catalyst.
In the ususal procedure employed, the glucosone is
hydrogenated at elevated pressure and temperature over the
selected metal catalyst until the desired degree of hydrogenation
30has been achieved. Pressures can range from 100 to 700 at~os-
phere and even higher while the temperature can range up to
about 200C. Pre~erred is 100 to 150C. and a pressure of
about 500 atmospheres.
The following example further illustrates the
35invention.
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1 EXAMPLE
~Iycelium of O. mucida are ~rown in accordance with
Example 1 of Czechoslovakian patent 175897 and the equivalent
of 15 g. (dry weight) of the mycelium is suspended in 3 L.
5Of 2.5% glucose solution 0.05M NaF in one zone of a 10 L.
reactor fitted with a hydrogen peroxide-permeable membrane
to form two zones. In the second zone, catalase enzyme immo-
bilized Oll DEAE-cellulose (Cellex- ~ manufactured by Bio-Rad
Laboratories~ is suspended in 3 L. of water.
The suspension in the first zone is mixed at 25~C.
and aerated with oxygen while the second zone is also mixed.
After 24 hours the mycelium is then separated from the solu-
tion in the first zone and the resulting clear solution is then
hydrogenated over Raney Ni at 500 atmospheres hydrogen gas and
5 100C. The aqueous mixture is filtered clear of the catalyst,
decolorized with carbon, dionized with ion-exchange (anionic
and cationic), and concentrated to a fructose syrup at reduced
pressure. Alternatively, the aqueous mixture is concentrated
and fructose allowed to crystallize.
The fructose obtained as either syrup or crystalline
product is of food grade quality.
The membrane employed in this example permits passage
- of molecules of molecular weight of less than 50.
Essentially the same results are obtained when
25 O. mucida is replaced with the following organisms:
Polyporus obtusus
Radulum casearium
Lenzites Trabea
Irpex flanus
Polyporus versicolor
Pellicularia filamentosa
Armillaria mellea
Schizophyleum commune
Corticium caeruleum
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