U.S. patent application number 10/383891 was filed with the patent office on 2003-10-23 for methods of separating a corn fiber lipid fraction from corn fiber.
This patent application is currently assigned to Eastman Chemical Company. Invention is credited to Buchanan, Charles M., Buchanan, Norma L., Debenham, John S., Shelton, Michael C., Wood, Matthew D..
Application Number | 20030199087 10/383891 |
Document ID | / |
Family ID | 22384216 |
Filed Date | 2003-10-23 |
United States Patent
Application |
20030199087 |
Kind Code |
A1 |
Buchanan, Charles M. ; et
al. |
October 23, 2003 |
Methods of separating a corn fiber lipid fraction from corn
fiber
Abstract
In one aspect, the invention provides a method of separating a
corn fiber lipid fraction having phytosterol esters and
phytosterols wherein the method comprises the steps of: (a)
providing a mixture of unground corn fiber and water; (b)
separating the liquid from the corn fiber, thereby providing a
water wet corn fiber; and (c) extracting the water wet corn fiber
with at least one polar organic solvent, thereby providing a corn
fiber lipid fraction/polar organic solvent solution having
phytosterol esters and phytosterols.
Inventors: |
Buchanan, Charles M.;
(Kingsport, TN) ; Buchanan, Norma L.; (Kingsport,
TN) ; Debenham, John S.; (Kingsport, TN) ;
Shelton, Michael C.; (Kingsport, TN) ; Wood, Matthew
D.; (Gray, TN) |
Correspondence
Address: |
NEEDLE & ROSENBERG P C
127 PEACHTREE STREET N E
ATLANTA
GA
30303-1811
US
|
Assignee: |
Eastman Chemical Company
|
Family ID: |
22384216 |
Appl. No.: |
10/383891 |
Filed: |
March 6, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10383891 |
Mar 6, 2003 |
|
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09501917 |
Feb 10, 2000 |
|
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60119399 |
Feb 10, 1999 |
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Current U.S.
Class: |
435/412 |
Current CPC
Class: |
C08B 3/00 20130101; C13K
1/08 20130101; C13K 1/02 20130101; A23L 7/107 20160801; C08B 30/10
20130101; A23V 2002/00 20130101; D21C 5/005 20130101; A23K 20/163
20160501; D21C 5/00 20130101; C08B 37/006 20130101; A23K 20/147
20160501; C13K 13/00 20130101; C08B 37/0057 20130101; C11B 1/10
20130101; A23K 10/37 20160501; A23L 33/115 20160801; C13K 13/002
20130101; Y02P 60/87 20151101; A23L 7/115 20160801; A23L 29/262
20160801; A23L 33/11 20160801; A23L 33/24 20160801; Y02P 60/877
20151101; C08B 11/00 20130101; C11B 1/025 20130101; A23D 9/007
20130101; A23D 9/02 20130101; A23V 2002/00 20130101; A23V 2250/602
20130101; A23V 2002/00 20130101; A23V 2250/638 20130101; A23V
2002/00 20130101; A23V 2250/626 20130101 |
Class at
Publication: |
435/412 |
International
Class: |
C12N 005/04 |
Claims
What is claimed is:
1. A method of separating a corn fiber lipid fraction having
phytosterol esters and phytosterols wherein the method comprises
the steps of: a. providing a mixture of unground corn fiber and
water; b. separating the liquid from the corn fiber, thereby
providing a water wet corn fiber; and c. extracting the water wet
corn fiber with at least one polar organic solvent, thereby
providing a corn fiber lipid fraction/polar organic solvent
solution having phytosterol esters and phytosterols.
2. The method of claim 1, wherein the organic solvent comprises
methanol, ethanol, isopropyl alcohol, n-butyl alcohol, acetone,
ethyl acetate, methyl isobutyl ketone, methyl ethyl ketone, or a
mixture thereof.
3. The method of claim 1, wherein the organic solvent comprises
isopropyl alcohol, acetone, ethyl acetate, or a mixture
thereof.
4. The method of claim 1, wherein the solvent extraction step is
conducted at a temperature of from about 0.degree. C. to about
115.degree. C. for from about 1 to about 600 min.
5. The method of claim 1, wherein the solvent extraction step is
conducted at a temperature of from about 20.degree. C. to about
80.degree. C. for from about 10 to about 60 min.
6. The method of claim 1, wherein the water wet corn fiber contains
from about 15% to about 85% water based on dry weight of the corn
fiber.
7. The method of claim 1, wherein the water wet corn fiber contains
from about 25% to about 65% water based on dry weight of the corn
fiber.
8. The method of claim 1, further comprising separating the corn
fiber lipid fraction from the organic solvent solution wherein the
phytosterol and phytosterol esters are separated with the corn
fiber lipid fraction.
9. The method of claim 8, further comprising separating phytosterol
esters and phytosterols from the corn fiber lipid fraction and,
optionally, separating the phytosterol esters and phytosterols from
each other.
Description
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application is a divisional of, and claims the benefit
of, application Ser. No. 09/501,917, filed on Feb. 10, 2000, which
status is allowed. The Ser. No. 09/501,917 application claims
priority to U.S. Provisional Application Serial No. 60/119,399,
filed Feb. 10, 1999. U.S. application Ser. Nos. 09/501,917 and
60/119,399 are each incorporated herein by this reference in their
entireties.
FIELD OF THE INVENTION
[0002] This invention relates to corn fiber. More particularly,
this invention relates to the extraction of a corn fiber oil from
wet or dry milled corn fiber. Specifically, the corn fiber obtained
according to the methods herein comprises phytosterols and
phytosterols esters. The invention also relates to novel corn fiber
oil obtained from corn fiber.
BACKGROUND OF THE INVENTION
[0003] In the future, it will become increasingly important to
develop consumer products from renewable resources, especially from
annually renewable resources. Corn is one example of an annually
renewable resource that serves as a source of valuable consumer
products. Products derived from corn serve an important role in
providing useful foodstuffs to the public. Corn provides important
products, such as high fructose corn syrup, ethanol, grain and corn
oil. While a large percentage of the total portion of corn is
utilized to manufacture these substances, as well as other high
value products, a significant fraction of corn is utilized for
relatively low value products, such as animal feed. Technology that
would allow a higher value utilization of the remaining fractions
of corn would provide increased value overall from the entire
useable portions of corn.
[0004] Corn fiber is one under-utilized fraction of corn. Corn
fiber is obtained as a major by-product of the milling of corn.
Corn fiber comprises the outer hull portion of the corn kernel. It
has been estimated that approximately 7 to 10 billion pounds of
corn fiber are produced annually in the United States. The fiber is
produced at milling facilities and is collected as a relatively
homogeneous fraction.
[0005] A major source of corn fiber is the wet milling of corn.
During this process, the higher value products are removed from
corn, such as the germ of the kernel. After extraction of the high
value products, the remainder, which generally constitutes corn
fiber, is mixed with steep liquor, also a by-product of corn
milling. The mixture of fiber and liquor is then normally dried,
pelletized and sold as gluten.
[0006] Another source of corn fiber is dry milling; corn fiber
obtained from dry milling is often referred to as "corn bran." The
bran by-product of dry milled corn fiber, composed primarily of
hull, is mixed with other corn by-products and sold as hominy.
[0007] Both gluten feed and hominy feed are fairly low-value
products. Nonetheless, these products have generally been the only
commercial products prepared from corn fiber. Given the low margins
of such products made from corn fiber, it is not uncommon for corn
fiber to be disposed of outright, instead of undertaking the effort
to prepare such low-value products.
[0008] Corn fiber makes up a significant (5 to 10 wt. %) portion of
the total weight of the corn kernel. Corn fiber itself is made up
of a number of components most of which, if extracted, can be
commercially valuable. Specifically, corn fiber consists primarily
of residual starch (10 to 25 wt. %), hemicellulose (40 to 50 wt.
%), cellulose (15 to 25 wt. %), phenolic acids (3 to 5%), with the
remainder present as proteins and oils. (See Wolf, et al. Cereal
Chemistry, 30(1953), pp. 195-203; Chanliaud, et al., J. Cereal
Science, 21(1995), pp. 195-203.) The variations in the reported
composition are believed to be due to corn plant variety and growth
conditions, as well as the specific methods utilized to isolate the
corn fiber.
[0009] Hemicellulose is a component of corn fiber that has been of
interest commercially. A number of references disclose the
extraction of hemicellulose from corn fiber. However, most previous
attempts to obtain useful products from corn fiber have focused
almost entirely on methods to extract hemicellulose from corn fiber
and the properties, particularly the color, of the hemicellulose
obtained. These attempts to extract hemicellulose were likely
initiated by the fact that hemicellulose has several valuable
properties that make it attractive for a number of applications. In
a non-exclusive list, some uses for hemicellulose include non-toxic
adhesives, thickeners, emulsifiers, stabilizers, film formers and
paper additives. (See e.g., Whistler, Industrial Gums, 3d Ed.,
Academic Press, 1993, pp. 295-308; U.S. Pat. No. 2,772,981; Wolf,
et al. Cereal Chemistry, 30(1953), pp. 451-470.)
[0010] As indicated by these, as well as other references,
hemicellulose can be quite difficult to extract from corn fiber.
Because corn fiber hemicellulose is soluble in H.sub.2O, it would
be expected that hemicellulose would be fairly easy to extract from
corn fiber utilizing water or some other non-aggressive solvent.
This is not the case, however. Hydrogen bonding and physical
entanglement of the hemicellulose with the corn fiber matrix are
believed to be in part responsible for the difficulty in
extraction. Other reasons for the difficulty in extractability may
be due to cross-linking of the hemicellulose to other components of
the corn fiber cell wall via covalent bonds between esterified
phenolic acid residues and arabinose residues.
Protein-polysaccharide linkages may also affect the ability to
extract hemicellulose from corn fiber.
[0011] Most previous attempts to extract hemicellulose from corn
fiber have focused on the use of strongly alkaline materials. This
is not surprising, as one definition of hemicellulose is the
portion of plants that is extractable by hot alkali treatment.
[0012] Various references disclose techniques to extract
hemicellulose. For example, U.S. Pat. No. 2,709,699 discloses
extraction of corn fiber with aqueous alkali at a pH of from 9 to
13 at from 90 to 115.degree. C. for a time sufficient to solubilize
hemicellulose so that it can be extracted. In this reference, the
hemicellulose was isolated by adjusting the solution pH with an
inorganic acid, followed by precipitation of the hemicellulose in
ethanol, filtering to remove the hemicellulose and drying.
[0013] In another reference disclosing the extraction of
hemicellulose from corn fiber, U.S. Pat. No. 4,038,481, corn fiber
is treated with alkali to solubilize the hemicellulose. The
hemicellulose is then precipitated with a water miscible organic
solvent. The solvents utilized are acetone, methanol, ethanol,
propanol, isopropanol, isobutyl alcohol, tert-butyl alcohol, or a
mixture thereof. There is no disclosure of precipitation with
acetic acid in this reference.
[0014] A recent reference, W098/40413, discloses extraction of
hemicellulose by heating corn fiber with alkaline hydrogen
peroxide; the peroxide may be added at the same time or after an
alkaline material, such as NaOH or Ca(OH).sub.2, is added.
Significantly, W098/40413 discloses the hemicellulose extractant as
being heated in the presence of the alkaline hydrogen peroxide in
order to obtain a suitably white hemicellulose powder from the
precipitation step. However, this method is exceedingly dangerous
to practice on an industrial scale because of excessive emissions
of gas which may lead to significant foaming of the strongly
alkaline materials and possibly to explosions.
[0015] Furthermore, although hemicellulose itself is a valuable
product, the sub-components of hemicellulose are of even higher
value. No reference has been located which addresses the extraction
of these valuable sub-components from corn fiber. With the
invention herein, it has been found that hemicellulose obtained
from corn fiber may be subjected to further processing to provide
carbohydrate fractions of very high value. That is, in accordance
with the invention herein, it has been found possible to extract a
number of valuable monosaccharide materials from corn fiber. Also
in accordance with the invention herein, it has been found that
hemicellulose from corn fiber may be derivatized to form corn fiber
arabinoxylan esters and ethers. Methods of processing corn fiber
hemicellulose in such a manner are not believed to be disclosed in
the prior art.
[0016] Other than to obtain hemicellulose, there have been few
attempts to exploit the remaining components of corn fiber. A
notable recent exception relates to corn fiber oil. Corn fiber oil
contains a significant portion of plant sterol esters. These
materials have been reported to be useful as nutraceuticals,
particularly as hypocholesterolemics. At this time, rice bran oil
and tall oil are the major source of plant sterol esters utilized
for commercial purposes.
[0017] Rice bran has been reported to contain approximately 18 wt.
% extractable oil. Of this amount, 0.1 to about 0.8 wt. % comprises
a ferulate ester, meaning that rice bran, at most, contains only
about 0.08 wt. % ferulate ester. Moreover, the phytosterol esters
in rice bran oil are primarily gamma-oryzanols, which are believed
to be less effective as hypocholesterolemics.
[0018] In contrast, corn fiber oil has been shown to contain
approximately 0.54 to 3.5 wt. % extractable oil and, of this, about
6.75 wt. % is a ferulate ester. Corn fiber therefore comprises
about 0.12 wt. % ferulate ester, a significantly higher amount of
ferulate ester than is present in the most commercially utilized
source of hypocholesterolemic oils which are obtained from rice
bran.
[0019] A recent patent, U.S. Pat. No. 5,843,499, discloses the
extraction of corn fiber oil from finely ground corn fiber by
utilizing either hexane or supercritical CO.sub.2 as a solvent,
with hexane being preferred. In this reference, the degree of
grinding was demonstrated to be critical in determining the amount
of oil obtained from the corn fiber, with a finer grinding of the
corn fiber resulting in a greater amount of oil extracted. Drying
of the corn fiber was also found to be highly significant to the
invention, presumably because when the corn fiber is wet, the
hexane extractant will not adequately penetrate the fiber so as to
allow satisfactory extraction. However, because a drying step is
expensive and time consuming on an industrial scale, it would be
highly beneficial to be able to extract phytosterol esters from
corn fiber directly without the need for an additional drying
step.
[0020] As noted, cellulose forms a significant portion of corn
fiber. However, cellulose has not been isolated from corn fiber in
a form suitable for derivatization into higher value products, such
as cellulose esters and cellulose ethers. This is not surprising
because the prior art indicates that high purity cellulose was not
obtained from the previously utilized methods. For example, U.S.
Pat. No. 4,038,481, discussed previously, discloses that the
cellulose obtained according to the methods therein contained about
35 wt. % contaminates which were believed to be present in the
forth of insoluble hemicellulose. This contamination would make it
difficult, if not impossible, to utilize the cellulose obtained
according to the methods of the '481 patent for preparation of
cellulose derivatives.
[0021] In summary, no known reference addresses methods to obtain
maximum utilization of the various components of corn fiber.
Instead, the references have focused specifically on the
optimization of hemicellulose color, and, separately, on methods to
extract oil from corn fiber. While these are valuable objectives in
and of themselves, in order to make the use of corn fiber an
economically viable process, it is necessary to utilize as many
components of corn fiber as possible. Furthermore, it is necessary
to develop methods to separate each of these valuable components
individually while leaving the remainder of the corn fiber so that
the further components can be efficiently extracted in order to
maximize value.
SUMMARY OF THE INVENTION
[0022] In one aspect, the invention provides a method of separating
a corn fiber lipid fraction having phytosterol esters and
phytosterols wherein the method comprises the steps of: (a)
providing a mixture of corn fiber and water; (b) contacting the
mixture with a protease enzyme to provide a proteolyzed corn fiber
and a liquid; (c) separating the liquid from the proteolyzed corn
fiber; and (d) extracting the proteolyzed corn fiber with at least
one organic solvent, thereby providing a corn fiber lipid
fraction/organic solvent solution having phytosterol esters and
phytosterols.
[0023] In a further aspect, the invention provides a method of
separating from corn fiber a lipid fraction having phytosterol
esters and phytosterols wherein the method comprises the steps of:
(a) heating an aqueous mixture of unground corn fiber; (b)
contacting the mixture of step (a) with at least one enzyme
suitable for digesting starch for a time and at a temperature
suitable to provide a mixture of an essentially destarched corn
fiber and a liquid comprising soluble carbohydrates; (c) contacting
the mixture of step (a) or (b) with a protease enzyme to provide a
proteolyzed corn fiber and a liquid; (d) separating the liquid of
step (c) from the corn fiber to provide a destarched, proteolyzed
corn fiber; and (e) extracting the destarched, proteolyzed corn
fiber with at least one organic solvent, thereby providing a corn
fiber lipid fraction/organic solvent solution having phytosterol
esters and phytosterols.
[0024] In yet a further embodiment, the invention provides a method
of separating a corn fiber lipid fraction having phytosterol esters
and phytosterols wherein the method comprises the steps of: (a)
providing a mixture of unground corn fiber and water; (b)
separating the liquid from the corn fiber, thereby providing a
water wet corn fiber; and
[0025] extracting the water wet corn fiber with at least one polar
organic solvent, thereby providing a corn fiber lipid
fraction/polar organic solvent solution having phytosterol esters
and phytosterols.
[0026] In a still further embodiment, the invention provides a corn
fiber lipid fraction containing phytosterols and phytosterols
esters obtained via solvent extraction of a proteolyzed corn fiber,
wherein the concentration of phytosterols in the lipid fraction is
at least about 1.4 times greater than the concentration of
phytosterols in the lipid fraction of a nonproteolyzed corn
fiber.
[0027] In a further aspect, the invention provides a corn fiber
lipid fraction containing phytosterols and phytosterol esters
obtained via solvent extraction of a proteolyzed corn fiber,
wherein the concentration of phytosterol esters in the lipid
fraction is at least about 1.4 times less than the concentration of
phytosterol esters in the lipid fraction of a nonproteolyzed corn
fiber.
[0028] Still further, the invention provides a method of obtaining
soluble proteins and carbohydrates from corn fiber wherein the
method comprises the steps of: (a)
[0029] a. heating an aqueous suspension of corn fiber; (b)
contacting the fiber sequentially or concurrently with an amylase
enzyme and protease enzyme for a time and at a temperature
sufficient to provide an essentially destarched, proteolyzed corn
fiber and a liquid comprising soluble proteins and carbohydrates;
and (c) separating the liquid from the destarched corn fiber
wherein the soluble proteins and carbohydrates are suitable as
feedstock for the production of animal feed, chemicals, and
polymers.
[0030] b.
[0031] Yet still further, the invention provides a method for
obtaining animal feed wherein the method comprises the steps of:
(a) heating an aqueous suspension of corn fiber; (b) contacting the
fiber sequentially or concurrently with an amylase enzyme and
protease enzyme for a time and at a temperature sufficient to
provide an essentially destarched, proteolyzed corn fiber and a
liquid comprising soluble proteins and carbohydrates; (c)
separating the liquid from the destarched, proteolyzed corn fiber;
(d) contacting the corn fiber at least once with an alkaline
extractant to provide an insoluble cellulose material and a liquid
comprising arabinoxylan; (e) separating the insoluble cellulose
material from the liquid comprising arabinoxylan; (f) adding a
sufficient amount of a corn steep liquor to the cellulose material;
and (g) removing water from the heterogeneous mixture, thereby
providing an animal feed.
[0032] Additional advantages of the invention will be set forth in
part in the description that follows, and in part will be obvious
from the description, or may be learned by practice of the
invention. The advantages of the invention will be realized and
attained by means of the elements and combinations particularly
pointed out in the appended claims. It is to be understood that
both the foregoing general description and the following detailed
description are exemplary and explanatory only and are not
restrictive of the invention, as claimed.
[0033] The accompanying drawings, which are incorporated in and
constitute a part of this specification, illustrate several
embodiments of the invention and together with the description,
serve to explain the principles of the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0034] FIG. 1 shows the corn fiber destarching rate where the data
has been normalized relative to the total sugars extracted.
[0035] FIG. 2 shows the initial corn fiber destarching rate where
the data has been normalized relative to total sugars
extracted.
[0036] FIG. 3 shows the Carbon 13 NMR spectra of phytosterol ester
isolated from destarched corn fiber oil.
[0037] FIG. 4 shows the caustic extraction rates of destarched corn
fiber measured in situ where the solubilized arabinoxylan has been
normalized to total extractables.
[0038] FIG. 5 shows the isolated yield of hemicellulose for each
extraction step in a continuous extraction of destarched corn
fiber.
[0039] FIG. 6 is a photomicrograph of small particles that can
often be present in liquids after caustic extraction of destarched
corn fiber.
[0040] FIG. 7 shows a HPLC analysis of corn fiber oil obtained by
(a) hexane extraction of dry, unground, untreated corn fiber; and
(b) hexane extraction of dry, unground, amylase and protease
treated corn fiber
[0041] FIG. 8 shows a proton NMR spectrum of arabinoxylan and
arabinoxylan methyl ether prepared from arabinoxylan.
[0042] FIG. 9 shows a proton NMR spectrum of the product obtained
after 6.25 hours of hydrolysis of arabinoxylan at 100.degree.
C.
[0043] FIG. 10 shows a carbon 13 spectrum of a selective hydrolysis
of an arabinoxylan series in which the most important resonances
corresponding to either xylose or arabinose have been labelled.
[0044] FIG. 11 shows a proton NMR spectra of arabinose obtained by
crystallization of an arabinoxylan hydrolysis sample, wherein the
sample is compared to that for a known arabinoxylan standard.
[0045] FIG. 12 shows pulse test results for a L-arabinose/xylose
separation experiment.
[0046] FIG. 13 shows pulse test results for a corn fiber
xylose/arabinose separation.
[0047] FIG. 14 shows pulse test results from an arabinose/ribose
separation experiment.
[0048] FIG. 15 shows pulse test results for xylose/arabinose
separation using a strongly anion exchange resin in the phosphate
form.
DETAILED DESCRIPTION OF THE INVENTION
[0049] The present invention may be understood more readily by
reference to the following detailed description of preferred
embodiments of the invention and the Examples included herein and
to the Figures and their previous and following descriptions.
[0050] It must be noted that, as used in the specification and the
appended claims, the singular forms "a," "an" and "the" include
plural referents unless the context clearly dictates otherwise.
Thus, for example, reference to "an aromatic compound" includes
mixtures of aromatic compounds, reference to "a carrier" includes
mixtures of two or more such carriers, and the like.
[0051] Ranges are often expressed herein as from "about" one
particular value, and/or to "about" another particular value. When
such a range is expressed, another embodiment includes from the one
particular value and/or to the other particular value. Similarly,
when values are expressed as approximations, by use of the
antecedent "about," it will be understood that the particular value
forms another embodiment.
[0052] A weight percent of a component, unless specifically stated
to the contrary, is based on the total weight of the formulation or
composition in which the component is included. Further, unless
otherwise noted, weight percents are expressed as dry weight. For
example, when a component is expressed as a weight percent based
upon corn fiber, the weight is based upon dry weight of corn
fiber.
[0053] Throughout this publication, where publications are
referenced, the disclosures of these publications in their
entireties are hereby incorporated by reference into this
application in order to more fully describe the state of the art to
which this invention pertains.
[0054] Unless otherwise noted, the percent carbohydrate has been
normalized to 100%. Abbreviations used herein or as follows: "Glu"
is glucose; "Xyl" is xylose; "Gal" galactose; "Ara" is arabinose;
"Man" is mannose, "DS" is the degree of substitution per monomer
unit, "EtOH" means ethanol, "MeOH" means methanol, "HPLC" means
high pressure liquid chromatography, "HOAc" or "AcOH" mean acetic
acid and "nd" means not determined. The term "DS/CU" means Average
Degree of Substitution/Carbohydrate Unit. The term "MS/CU" means
Average Molar Substitution/Carbohydrate Unit.
[0055] "Proteolyzed" means that the corn fiber has been treated
with a protease enzyme. "Nonproteolyzed" means that the corn fiber
has not been treated with a protease enzyme.
[0056] Hours is abbreviated as "h;" minutes are abbreviated as
"min."
[0057] The term "arabinoxylan" means that material extracted from
corn fiber after treatment with an alkaline extractant. Unless
otherwise indicated, arabinoxylan obtained according to the methods
herein may be comprised of either hemicellulose B or hemicellulose
A or a mixture thereof. In some instances, which will be evident to
one of ordinary skill in the art, the term "hemicellulose" is used
interchangeably with the term "arabinoxylan."
[0058] In a first major embodiment, the invention pertains to the
separation of a corn fiber lipid fraction having phytosterol esters
and phytosterols wherein the method comprises the steps of: a)
heating an aqueous mixture of unground corn fiber; b) contacting
the mixture of step (a) with at least one enzyme suitable for
digesting starch for a time and at a temperature suitable to
provide a mixture of an essentially destarched corn fiber and a
liquid comprising soluble carbohydrates; c) contacting the mixture
of step (a) or (b) with a protease enzyme to provide a proteolyzed
corn fiber and a liquid; d) separating the liquid of step (c) from
the corn fiber, thereby providing destarched, proteolyzed corn
fiber; and e) extracting the destarched, proteolyzed corn fiber
with at least one organic solvent, thereby providing a corn fiber
lipid fraction/organic solvent solution having phytosterol esters
and phytosterols.
[0059] In a further preferred embodiment, the invention pertains to
the separation of a novel corn fiber lipid fraction having
phytosterol esters and phytosterols wherein the method comprises
the steps of: a) providing a mixture of corn fiber and water; b)
contacting the mixture with a protease enzyme to provide a
proteolyzed corn fiber and a liquid; c) separating the liquid from
the proteolyzed corn fiber; and d) extracting the proteolyzed corn
fiber with at least one organic solvent, thereby providing a corn
fiber lipid fraction/organic solvent solution having phytosterol
esters and phytosterols.
[0060] Corn fiber generally comprises the outer portion of a corn
kernel. Thus, in one embodiment, the invention herein allows that
portion of corn that would normally be thrown away or
under-utilized as a low-value product to be further processed to
provide a number of high value materials.
[0061] In a surprising discovery, with the invention herein, it is
particularly preferable to utilize corn fiber containing SO.sub.2.
That is, it has been determined that corn fiber containing SO.sub.2
may be destarched as rapidly or more rapidly than corn fiber not
containing SO.sub.2. This result is unexpected because it would be
thought that SO.sub.2 would act as a biocide and thus retard the
enzymatic degradation of starch. This surprising discovery allows
utilization of corn fiber without the expense and difficulty of
drying the corn fiber prior to enzymatic treatment. Furthermore, in
some circumstances, drying the corn fiber can change the morphology
of the corn fiber thereby making it more difficult to remove the
starch and other components.
[0062] Since SO.sub.2 is an artifact of the wet milling process, in
a particularly preferred embodiment, the corn fiber is obtained
from a wet milling process. In another, still preferred embodiment
the corn fiber is obtained from a dry milling process. In another
embodiment, the corn fiber may generally be utilized from the
supplier "as-is" i.e., with no additional processing, wherein
SO.sub.2 is present in the corn fiber. In a preferred embodiment,
the amount of SO.sub.2 in the corn fiber is from about greater than
0 to about 5.0% by weight. In a further preferred embodiment, there
is no SO.sub.2 in the corn fiber. Still further, SO.sub.2 may be
added to either wet milled or dry milled corn fiber. In another
preferred embodiment, the corn fiber may be a mixture of corn fiber
obtained from both a wet milling and a dry milling process. Corn
fiber from a wet milling process can be obtained from Staley, Ill.
Corn fiber from a dry milling process can be obtained from the
Lauhoff Grain Company (Danville, Ill.).
[0063] One of ordinary skill in the art would recognize the
processing steps involved in wet milling and dry milling corn.
Generally, the corn to be wet milled is first cleaned and steeped
with slightly acidic water containing SO.sub.2. The kernels are
then coarsely milled and separated into constituents such as germ,
gluten and starch. The individual components are washed and dried
to produce end products such as corn starch, corn sweetener, corn
oil and animal feed by-products. In a dry milling process, corn
kernels are ground and separated into corn bran without a steeping
step. Corn fiber would normally constitute by-products of each of
these processing methods.
[0064] In a preferred embodiment, the corn fiber utilized in the
processes herein is unground. As used herein, "unground" means that
the fiber is used as obtained; that is, the fiber is not subjected
to a separate grinding step. It should be noted, however, that when
the corn fiber is obtained as a by-product of corn milling, such as
from a corn wet milling process, the corn fiber may not be whole.
Rather, since the fiber is subjected to processing steps in
conjunction with the wet milling and dry milling processes, such
"unground" corn fiber may have a wide distribution of particle
sizes. In a further embodiment, the corn fiber is subjected to a
separate corn fiber grinding step prior to being subjected to the
processing methods herein.
[0065] In one embodiment, the corn fiber is first heated. It is
generally believed that the corn fiber swells during the heating
step which improves the efficiency of the enzyme. The heating step
may be conducted at from about 50.degree. C. to about 120.degree.
C. for from about 1 to about 600 minutes or, in a further preferred
embodiment, the heating step is conducted at a temperature of from
about 50.degree. C. to about 100.degree. C. for about 1 to about 60
minutes. One of ordinary skill in the art will recognize that the
higher the temperature utilized, the shorter the time required to
provide a suitably swollen fiber. One of ordinary skill in the art
will also recognize that the optimum time for the heating step
depends upon the size and type of reactor vessel utilized. A
particularly preferred processing range for the heating/swelling is
from about 70.degree. C. to about 90.degree. C. for about 15 to
about 30 minutes.
[0066] In yet a further embodiment, the corn fiber is destarched
with an enzyme suitable for digesting starch. When the corn fiber
is to be subjected to both a destarching step and a proteolysis
step, the destarching may be conducted prior to or concurrently
with a protease enzyme contacting step, thereby providing an
essentially destarched and proteolyzed corn fiber. In a preferred
embodiment, the destarching is conducted utilizing an amylase
enzyme. In a most preferred embodiment, the destarching enzyme
comprises a thermophilic amylase. One example of a suitable
thermophilic amylase is marketed as "Spezyme." Spezyme is a
thermophilic .alpha.-amylase obtained from a genetically modified
strain of Baccillus lichenformis and is available from Genencor,
Palo Alto, Calif.
[0067] In a preferred embodiment, the destarching step is conducted
at a temperature of from about 25.degree. C. to about 120.degree.
C. for about 0.1 h to about 24 h at a pH of from about 4 to about
9. In a second, still preferred embodiment, the destarching step is
conducted at a temperature of from about 60.degree. C. to about
90.degree. C. for from about 1 h to about 12 h at a pH of from
about 7 to about 9. In some cases, depending upon the length and
temperature of the enzyme treatments, the time required to bring
the reaction mixture to the preferred temperature will also serve
to adequately swell the corn fiber. In a further embodiment, the
corn fiber may be destarched concurrently with the heating
step.
[0068] In one embodiment, the rate of destarching is followed by
utilizing a classical technique consisting of staining the corn
fiber with an iodine solution, wherein the solution is prepared by
adding iodine to H.sub.2O to provide about a 10% wt. solution. In
this method, a portion of the iodine solution is added to a small
vial containing a sample of corn fiber which has been subjected to
a destarching treatment. The point at which the corn fiber does not
stain purple generally indicates a complete or almost complete
destarching of the corn fiber.
[0069] In another embodiment, the rate of destarching is followed
in situ using an IR spectrophotometer (React IR 1000, ASI Applied
Systems, Millersville, Md.), wherein the enzymatic degradation of
starch is monitored by following the appearance of soluble
oligiosaccharides and monosaccharides in solution. The starch
digestion is judged to be complete when the concentration of
soluble species in solution no longer increases. This latter method
is particularly preferred in conjunction with the methods herein
because it permits real time monitoring of the destarching process
and is particularly well-suited for application in an industrial
process.
[0070] One of ordinary skill in the art will recognize that the
optimum temperatures, times and pH conditions for the destarching
step will vary due to the source of the specific enzyme utilized.
With the use of, for example, a thermophilic amylase enzyme such as
Spezyme, it has been surprisingly found that it is possible to
obtain a corn fiber which is at least about 90% destarched in less
than or about 20 minutes. This fairly rapid destarching makes the
methods disclosed and claimed herein particularly well-suited for
application on an industrial scale.
[0071] With the invention herein, in one particularly preferred
embodiment, it has been discovered that when it is desired to
obtain not only corn fiber oil, but also other materials such as
cellulose and arabinoxylan from corn fiber, it is critical to treat
the corn fiber with a protease enzyme prior to treatment of the
corn fiber with an alkaline extractant contacting step. The
protease enzyme contacting step of the invention herein also
provides novel products, as set out in more detail below.
[0072] Furthermore, it has also been unexpectedly found that
removal of a protein fraction of corn fiber by utilizing a protease
enzyme markedly increases the ability to filter the subsequently
alkaline extractant treated corn fiber. Moreover, in some
circumstances, treatment of corn fiber with a protease enzyme also
significantly reduces the quantity of alkaline material required
for extraction of arabinoxylan. For example, the prior art methods
disclose that a caustic strength of about 2.5 M was required to
efficiently extract the arabinoxylan from corn fiber not treated
with a protease. As set out in more detail below, as little as 0.5
M of caustic can be utilized to efficiently extract arabinoxylan
from proteolyzed corn fiber according to the methods herein. In one
particularly preferred embodiment, it is critical to contact the
corn fiber with a protease enzyme prior to performing any
subsequent processing steps, except for destarching and/or
heating/swelling of the fiber.
[0073] As one example of this particularly preferred process, the
time required to filter protease treated corn fiber was about 2
minutes, whereas the time required to filter a non-protease treated
corn fiber sample was about 12 hours. This dramatic decrease in
filtering time introduces marked efficiencies into the processes
herein and generally allows the undertaking of these processes on
an industrial scale.
[0074] Further, it has been unexpectedly found that treatment of
corn fiber with a protease enzyme provides for a more efficient
extraction of total corn fiber oil with a different distribution of
phytosterols and phytosterol esters than is observed with corn
fiber which has not been treated with a protease enzyme. Without
wishing to be bound by theory, it is believed that the protease
also functions as an esterase, thereby cleaving certain ester
linkages and allowing the more efficient extraction of the lipid
fraction from the corn fiber.
[0075] Significantly, it has been found that subjecting corn fiber
to a protease enzyme contacting step provides a corn fiber oil of a
different, and novel, composition over that corn fiber oil
disclosed in U.S. Pat. No. 5,843,499 to Moreau, et al.
Particularly, it has been found that corn fiber oil extracted from
proteolyzed corn fiber contains a different distribution of
phytosterols and phytosterol esters than the oil obtained by Moreau
et al. Accordingly, in one embodiment, the invention pertains to
the product obtained by the corn fiber oil extraction processes
herein. In yet another embodiment, the invention pertains to a
novel corn fiber oil in which the concentration of phytosterols in
the lipid fraction is at least about 1.4 times greater than the
concentration of phytosterols in the lipid fraction of
nonproteolyzed corn fiber. Still yet, the invention pertains to a
novel corn fiber oil in which the concentration of phytosterol
esters (or phytosterol ferulates) in the lipid fraction is at least
about 1.4 times less than the concentration of phytosterol esters
(or phytosterol ferulates) in the lipid fraction of nonproteolyzed
corn fiber.
[0076] A number of types of protease enzymes are useful in the
present invention. For example, Genencor (Palo Alto, Calif.)
supplies several types of proteases e.g., Purafect 4000L, Purafect
OX 4000L, Genencor Protease 899, Purafect OX-12000G, Purafect OxP
2000G, Purafect OxP 4000G, Purafect OXE and Protex 6L. These
proteases have wide ranges of activities under which they exhibit
maximum activity.
[0077] In a preferred embodiment, the protease enzyme contacting
step is conducted at a temperature of from about 25.degree. C. to
about 120.degree. C. for from about 0.1 h to about 24 h at a pH of
from about 4 to about 9. In a further embodiment, the protease
enzyme contacting step is conducted at a temperature of from about
60.degree. C. to about 90.degree. C. for from about 1 h to about 12
h at a pH of from about 7 to about 9. In a further embodiment, the
destarching step is conducted concurrently with the protease enzyme
contacting step.
[0078] One of ordinary skill in the art will recognize that
selection of reaction conditions for the enzymatic treatments
disclosed herein will be dependent upon the specific
characteristics of the enzyme utilized and will vary not only among
different suppliers, but also from lot to lot within the same
supplier. Thus, it is usually necessary to reassess the optimum
reaction conditions for the protease enzyme contacting step, as
well as any other enzymatic treatments utilized herein, upon
receipt of each new supply of enzyme. Suppliers normally provide
useful measurements of enzymatic activity from which the
appropriate reaction conditions may be determined.
[0079] When corn fiber oil is extracted, the oil extraction
techniques discussed herein may be utilized. However, it is
particularly preferred that unground, wet corn fiber be extracted
with a polar organic solvent to provide an excellent yield of corn
fiber oil. Thus, it is preferred that the corn fiber is not dried
(or is wet) prior to the organic solvent extraction step. The
inventors have surprisingly found that corn fiber may be extracted
while still wet from the prior treatments, such as any destarching
and proteolysis steps. In a preferred embodiment of this process,
the wet corn fiber is unground. One of ordinary skill in the art
would recognize that the omission of a drying step greatly improves
the industrial applicability of processes such as those herein. In
yet a further, still preferred, embodiment, the proteolyzed corn
fiber is dried prior to the solvent extracting step.
[0080] In a further preferred embodiment, the solvent utilized for
the extraction step is a polar organic solvent. In separate
embodiments, the polar organic solvent comprises methanol, ethanol,
isopropyl alcohol, n-butyl alcohol, acetone, ethyl acetate, methyl
isobutyl ketone, methyl ethyl ketone, or a mixture thereof. In a
separate, still preferred embodiment, the solvent comprises
acetone, isopropyl alcohol, ethyl acetate, or a mixture thereof. It
has surprisingly been found that use of a polar organic solvent, in
combination with wet, proteolyzed corn fiber, allows efficient
extraction of corn fiber oil from unground corn fiber. Thus, in a
particularly preferred embodiment, a polar solvent is utilized to
extract wet, proteolyzed corn fiber. In a further particularly
preferred embodiment, a polar solvent is utilized to extract wet,
proteolyzed and unground corn fiber. In a further preferred
embodiment, a polar solvent is utilized to extract wet,
non-proteolyzed and unground corn fiber.
[0081] In another, still preferred, embodiment the solvent utilized
to contract the corn fiber oil is a non-polar organic solvent.
Useful non-polar organic solvents comprise hexane, heptane, diethyl
ether, or a mixture thereof. When a polar organic solvent is used,
it is particularly preferred that the corn fiber is dry. In one
preferred embodiment, a non-polar solvent is utilized to extract
dry, unground corn fiber. In another preferred embodiment, a
non-polar solvent is utilized to extract unground corn fiber.
[0082] In another preferred embodiment, the solvent extraction step
is conducted at a temperature of from about 0.degree. C. to about
115.degree. C. for from about 1 mill to about 600 min. In a still
further preferred embodiment, the solvent extraction step is
conducted at a temperature of from about 20.degree. C. to about
80.degree. C. for from about 10 to about 60 min. Those skilled in
the art will recognize that the most preferred temperature will be
a temperature slightly below the boiling temperature of the solvent
utilized. Further, one of ordinary skill in the art would recognize
that a number of techniques can be utilized to extract the corn
fiber lipid fraction, including continuous extraction in which the
extraction liquid is permitted to flow over the fiber or where the
fiber flows counter-current to the extraction liquid, as well as
single-batch extractions where the extraction liquid is maintained
in contact with the fiber for sufficient time to solubilize the
soluble oils. It is particularly preferred that the corn fiber is
extracted utilizing a continuous process.
[0083] Following the solvent extraction step, the solvent is
generally removed from the corn fiber lipid fraction by
distillation. In the case were a polar organic solvent with limited
water solubility is used for extraction of wet corn fiber, any
water layer that may develop can be decanted either before or after
the distillation. An example of such an organic solvent with
limited water miscibility is EtOAc. When high vacuums (about 0.05
to about 10 mm Hg) and temperatures (about 50.degree. C. to about
200.degree. C.) are coupled with the appropriate equipment, such as
falling film or wiped film evaporators, the organic solvent can be
removed from the lipid fraction and the lipid fraction can be
enriched in phytosterols and phytosterol esters.
[0084] A preferred method for separating the phytosterols and
phytosterol esters is by crystallization. If desired, unwanted
components such as glyceride esters can be separated from the corn
fiber lipid fraction by extraction, thereby providing a lipid
fraction enriched in phytosterol esters and phytosterols from which
the phytosterol esters and phytosterols can be crystallized. In one
embodiment unwanted components in the corn fiber lipid fraction are
any components not consisting of phytosterols and/or phytosterol
esters. Yet further, unwanted components can be removed from the
corn fiber lipid fraction by hydrolysis of glyceride esters
followed by removal of the hydrolyzed compounds to provide a lipid
fraction enriched in phytosterol esters and phytosterols. The
phytosterol esters and phytosterols can be isolated from this
enriched lipid fraction by crystallization. One of ordinary skill
in the art will recognize the methods that may be utilized to
perform such separations.
[0085] As noted, the corn fiber lipid fraction is preferably
separated from the organic solvent solution. Preferably, the
organic solvent is separated from the corn fiber lipid fraction via
distillation. A preferred distillation method is vacuum
distillation. By this preferred vacuum distillation method, the
corn fiber lipid fraction may be enriched in phytosterols and
phytosterol esters. The corn fiber lipid fraction may be separated
from the organic solvent in a distillation process wherein the
phytosterols and phytosterol esters are separated with the corn
fiber lipid fraction. By "separated with" it is meant that the
phytosterols and phytosterol esters are present in the corn fiber
lipid fraction.
[0086] It is further preferable that the phytosterols and
phytosterol esters are separated from the corn fiber lipid fraction
in a mixture or as individual fractions. When the phytosterols and
phytosterol esters are separated as a mixture, the phytosterols may
optionally be separated from the phytosterol esters to provide two
fractions: one fraction consisting essentially of a phytosterol and
one fractoin consisting essentially of a phytosterol ester.
[0087] In a further preferred embodiment, prior to conducting the
distillation step, a water layer is separated from the corn fiber
lipid fraction/organic solvent solution. Still further, it is
preferred that after conducting the distillation step, a water
layer is separated from the corn fiber lipid fraction/organic
solvent solution.
[0088] In a further embodiment, the invention comprises obtaining a
yield of corn fiber oil of at least 1.0% as measured by dry weight
of corn fiber. In a preferred embodiment, the corn fiber oil is
obtained in a yield of about 1.0 wt. % to about 7.0 wt. % based on
the dry weight of corn fiber. In a still further preferred
embodiment, the corn fiber oil is obtained in a yield of about 3
wt. % to about 5 wt. % based on the dry weight of corn fiber. Still
further, the amount of corn fiber oil extracted is from about 2 wt.
% to about 3.5 wt. %. The methods herein provide enhanced yields of
total extractable corn fiber oil, as well as enhanced yields of
phytosterol esters and phytosterols in the corn fiber oil relative
to those disclosed in the prior art.
[0089] Yet another embodiment comprises conducting at least one
additional solvent extraction step, wherein the corn fiber lipid
fraction/organic solvent solution from a previous solvent
extraction step is reused in the additional solvent extracting
step, thereby providing an increasingly concentrated corn fiber
lipid fraction/organic solvent solution having phytosterols and
phytosterol esters. In this preferred embodiment, organic solvent
containing corn fiber oil is recycled for subsequent extractions.
The oil becomes increasingly more concentrated with each pass which
results in an overall higher concentration of corn fiber oil in the
final corn fiber oil/solvent solution. The number of additional
extraction steps may range from about 1 to about 100, more
preferably from about 2 to about 10. In a particularly preferred
embodiment, this solvent recycling step is utilized in conjunction
with a continuous extraction process. One of ordinary skill in the
art will recognize that such methods greatly improve the industrial
applicability of the extraction of corn fiber oil from corn
fiber.
[0090] In a further aspect, the invention provides a method of
extracting a corn fiber lipid fraction having phytosterols and
phytosterol esters wherein the method comprises the steps of: a)
providing a mixture of unground corn fiber and water; b) separating
the liquid from the corn fiber, thereby providing a water wet corn
fiber; and c) extracting the water wet corn fiber with at least one
polar organic solvent, thereby providing a corn fiber lipid
fraction/polar organic solvent solution having phytosterol esters
and phytosterols. In this preferred method, corn fiber utilized to
obtain corn fiber oil is not treated with a protease enzyme prior
to solvent extraction. Still further preferred, the corn fiber is
unground. Still preferably, the corn fiber is water wet, wherein
the water wet corn fiber contains from about 15 wt. % to about 85
wt. % water based on dry weight of the corn fiber. Yet further
preferred, the corn fiber is water wet, wherein the water wet corn
fiber contains from about 25 wt. % to about 65 wt. % water based on
dry weight of the corn fiber. Since the corn fiber in this
embodiment is wet, it is critical to utilize polar organic solvent
for the extraction.
[0091] With this embodiment, it has been found that when it is not
desired to extract additional materials from the corn fiber, it is
not necessary to conduct a proteolysis step. With the invention
herein it has been found that extraction of corn fiber oil alone
from non-proteolyzed corn fiber does not provide an increase in the
extraction efficiency of alkaline extraction. Thus, subsequent
processing steps, such as alkaline extraction, with this
non-proteolyzed corn fiber will be exceedingly slow, as discussed
above. Accordingly, this non-proteolyzed corn fiber utilized for
corn fiber oil extraction is generally unsuited for further use to
obtain other materials from corn fiber as discussed below.
[0092] In a next major aspect, the invention provides a method of
obtaining a cellulose material from corn fiber, wherein the method
comprises the steps: a) heating a mixture of corn fiber and a
liquid; b) contacting the mixture of step (a) with a protease
enzyme, thereby providing a proteolyzed corn fiber and a liquid; c)
separating the liquid from the proteolyzed corn fiber; d)
contacting the proteolyzed corn fiber at least once with an
alkaline extractant, thereby providing an insoluble cellulose
material and a first liquid comprising arabinoxylan; e) separating
the insoluble cellulose material from the first liquid comprising
arabinoxylan at a temperature of at or above about 60.degree. C.;
and f) rinsing the insoluble cellulose material to remove
essentially all alkali, thereby providing a cellulose material
having a glucose content of at least about 50% and consisting
essentially of cellulose I. In a particularly preferred embodiment,
the alkaline extractant does not comprise alkaline
H.sub.2O.sub.2.
[0093] In this embodiment, the corn fiber may first be treated by
any of the methods disclosed above. However, it is particularly
preferred, and even critical in some instances, to utilize a
proteolyzed corn fiber for the reasons set forth above. Further,
although it is not necessary in order to obtain suitable downstream
products, it is preferred that the corn fiber first be solvent
extracted to provide a corn fiber oil. One of ordinary skill in the
art will recognize that by extracting the corn fiber oil from the
fiber prior to alkaline extraction, the number of useful products
obtained from corn fiber may be maximized and, thus, the full
industrial applicability and value of the invention herein may be
realized.
[0094] In one embodiment of the alkaline extraction method, from
about 1 wt. % to about 50 wt. %, as measured by dry weight of corn
fiber, per 100% total volume of liquid is utilized in the alkaline
extractant contacting step. In a further embodiment, from about 10
to about 25 wt. %, as measured by dry weight of corn fiber per 100%
total volume of liquid is utilized in the alkaline extractant
contacting step. Still further, from about 5 wt. % to about 15 wt.
%, as measured by dry weight of corn fiber, per 100% total volume
of liquid is utilized in the alkaline extractant contacting step.
It has been surprisingly found that increasing the solids level
results in a lower .alpha. purity (% glucose) for the resulting
cellulose material. By increasing the alkali concentration, the
purity of the cellulose material can be increased. Thus, in a
preferred embodiment, when the amount of corn fiber solids in the
alkaline extraction step is high, a higher concentration of
alkaline extractant is utilized. In a particularly preferred
example, when concentration of NaOH is increased, the purity of the
cellulose material is increased.
[0095] In accordance with one embodiment of the invention herein,
the alkaline extractant comprises NaOH, KOH, Ca(OH).sub.2,
NH.sub.4OH, CaCO.sub.3, K.sub.2CO.sub.3, Na.sub.2CO.sub.3, LiOH, or
a mixture thereof at a concentration of from about 0.1 M to about
3.75 M. In a further preferred embodiment, the alkaline extractant
comprises NaOH, KOH, or a mixture thereof at a concentration of
from about 0.5 M to about 3.0 M, or from 0.5 M to about 2.0 M. Yet
still further preferably, the alkaline extractant comprises NaOH at
a concentration of from about 0.5 M to about 1.5 M. In a further
embodiment, the alkaline extractant contacting step is conducted at
from about 60.degree. C. to about 100.degree. C. In yet another
embodiment, one extractant contacting step is conducted for from
about 0.5 h to about 2 h, the alkaline extractant comprises NaOH,
KOH, or a mixture thereof at a temperature of from about 70.degree.
C. to about 90.degree. C. Still further preferably, the alkaline
extractant contacting step is conducted for less than 2 h, more
preferably, for from about 0.5 h to less than 2 h. Further
preferably, the alkaline extractant contacting step is conducted
for from about 0.5 h to about 1.5 h. Still further preferably, the
alkaline extractant contacting step is conducted for a time of from
about 1 second to about 4 hours. One of ordinary skill in the art
will recognize that the duration of each alkaline extractant
contacting step will vary in relation to at least the following
factors: a) the concentration of the alkaline material; b) the
temperature of the alkaline extractant contacting step; c) the
number of alkaline extractant contacting steps; d) the desired
purity of the cellulose material; and e) the number and type of any
additional processing steps.
[0096] Still preferably, the cellulose material is contacted with
the alkaline extractant one or more times. In a further, still
preferred embodiment, the cellulose material is contacted with the
alkaline extractant at least twice. Still further, the alkaline
extractant contacting step comprises at least three alkali
contacting steps wherein at each step the concentration of alkali
is decreased in relation to the step immediately preceding, thereby
providing a last alkaline contacting step wherein the pH of the
extractant is essentially neutral. In this latter method, the corn
fiber is first treated with a high concentration of alkaline
extractant, followed by at least one subsequent treatment of a
lower concentration alkaline extractant, whereby the final step
consists essentially of water wherein the pH of the water is
essentially neutral. This method is particularly preferable because
it has been found that if the initial concentration of alkaline
extractant in the contacting step is fairly high, e.g., greater
than about 1.5 M when the alkali is NaOH, most of the arabinoxylan
can be removed from the corn fiber to provide a liquid comprising
arabinoxylan. In a preferred embodiment, subsequent alkaline
extractions may be conducted to remove any residual arabinoxylan
from the cellulose material as discussed below.
[0097] It is preferred that at least the alkaline extractant
contacting step is conducted in a continuous process. However, it
is particularly preferable that all the steps according to the
processes herein are conducted in continuous processes in order to
increase the industrial applicability of the invention herein. In a
further embodiment the number of alkaline extractant contacting
steps is from about 2 to about 200. In yet a further preferred
embodiment, the cellulose material obtained from corn fiber is
contacted with the alkaline extractant from about 1 to about 100
times, still preferably, from about 5 to about 50 times, still
further preferably, the cellulose material is contacted with the
alkaline extractant from about 5 to about 25 times, or further
preferably, from 5 to 10 times.
[0098] One example of the preferred continuous process is a belt
filter process, wherein the proteolyzed corn fiber is applied to a
moving belt. In one embodiment, the alkaline extractant is applied
at the beginning of the belt while simultaneously applying a vacuum
to remove the alkaline extractant that makes up the liquid
comprising arabinoxylan. At the first end of the moving belt,
strong alkaline solutions are applied, with weaker solutions being
applied as the length of time on the belt increases. Under these
conditions, as the destarched corn fiber reaches a second end of
the moving belt, only hot (i.e., >60.degree. C.) water with an
essentially neutral pH is applied. Thus, the destarched corn fiber
is exposed to multiple contacting steps with diminishing alkaline
extractant strength, thereby providing an essentially
arabinoxylan-free cellulose material for further processing.
[0099] One of ordinary skill in the art will recognize that it may
be useful to collect the individual alkaline extractants when
multiple alkaline extractant contacting steps are conducted in
order to maximize the amount of arabinoxylan obtained. However, it
will also be recognized that the amount of extracted arabinoxylan
in the alkaline extractant may be low in subsequent extractions.
Thus, in accordance with the methods herein, subsequent alkaline
extractions with low or very low arabinoxylan concentrations may
not be collected with earlier alkaline extractants in order to
lessen the amount of liquid entering into the volume reducing step
that will, of course, need to be removed in the volume reducing
step.
[0100] In one particularly preferred embodiment relating to the
continuous process, a first alkaline extractant contacting step
comprises NaOH, KOH, or a mixture thereof at a concentration of
from about 0.1 M to about 1.5 M and a final alkaline extractant
contacting step consists essentially of water of water, wherein the
pH of the water is essentially neutral. Still further the
concentration of alkaline extractant in the preferred continuous
process is from 0.1 to about 3.75 M, still further, from about 0.1
to about 3.0 M, yet still further, from about 0.1 to about 2.0 M.
Moreover, using this preferred continuous process throughout the
invention herein, any subsequent treatment steps i.e., alkaline
H.sub.2O.sub.2 bleaching, acid rinsing or xylanase enzyme
treatment, as set out in more detail below, may be efficiently
conducted.
[0101] With the invention herein, it has been surprisingly
discovered that it is possible to obtain high purity cellulose from
corn fiber. In particular, it has been found possible to generate
cellulose suitable for derivatization utilizing the methods herein.
Accordingly, in a particularly preferred embodiment, the cellulose
material has a glucose content of above about 80% and consists
essentially of cellulose I.
[0102] With the invention herein, it has been found that if the
temperature of the alkaline extractant contacting step and any
subsequent contacting steps is allowed to fall below about
60.degree. C., a significant amount of the cellulose I will be
transformed into unreactive cellulose II. One of ordinary skill in
the art will recognize that derivatization of cellulose under
normal conditions will usually occur only if the cellulose material
is present as cellulose I. Thus, in order to obtain cellulose
material suitable for derivatization, it is critical with the
methods herein that the alkaline extractant contacting step takes
place at a temperature of at or above at least about 60.degree. C.
Further, if it is desired to utilize the cellulose material for
derivatization, it is critical to perform all subsequent steps,
including that of filtering the alkaline extractant (if in the
presence of the insoluble cellulose material), at a temperature at
or above 60.degree. C. in order to retain the morphology of the
cellulose material as cellulose I with a glucose content of equal
to or greater than about 80%. A preferred temperature range for
treatment of the cellulose material to preserve derivatizability is
from about 70.degree. C. to about 85.degree. C. One unexpected
benefit of processing the cellulose material at equal to or greater
than 60.degree. C. is that the viscosity of the arabinoxylan
extract (liquid comprising arabinoxylan and concentrated liquid) is
significantly reduced.
[0103] In one preferred embodiment of the invention herein, the
cellulose is at least 80% cellulose I and, more preferably, at
least 85% cellulose I, still more preferably 90%, and, even more
preferably, 95% cellulose I. As noted, it is preferable to wash the
cellulose fiber with sufficient water that is at or above about
60.degree. C. to remove most, if not all, of the alkaline
extractant before allowing the cellulose fiber to cool in order to
maintain the preferred cellulose I morphology. Optionally, the
water can contain about 0.1 wt. % to about 30 wt. % acetic acid or
other acid, which will serve to neutralize any residual alkaline
material. Further, in accordance with this invention, it is
preferred the cellulose material should not be allowed to cool
below about 60.degree. C. between the various steps for the reasons
noted above.
[0104] When cellulose I is not required, such as when the corn
fiber cellulose is intended for applications such as paper making,
it is not necessary that the steps subsequent to alkaline
extraction be conducted at a temperature of at or above about
60.degree. C. in order to maintain cellulose I morphology. This is
due to the fact that such cellulose need not be particularly
reactive and, accordingly may be of relatively unreactive cellulose
II morphology. In this case, the filtration of the alkaline
extractant can be conducted in the temperature range of from about
20.degree. C. to about 90.degree. C., with about 25.degree. C. to
about 50.degree. C. being the more preferred temperature range.
Also, for applications such as paper, the cellulose may have a
lower glucose content. A preferred range is from about 50% to about
80%, with from about 60% to about 75% being the more preferred
range. However, as noted, elevated temperatures will greatly aid in
reducing the solution viscosity of the arabinoxylan extract.
[0105] With the invention herein, it has been found possible to
further increase the purity of cellulose obtained by subjecting the
insoluble cellulose material to further treatments. In one
embodiment, it is preferable to first rinse the cellulose material
with sufficient water that is at a temperature of at or above about
60.degree. C. In a further, still preferred embodiment, the
cellulose material is not rinsed prior to subsequent
treatments.
[0106] In particular, it has been found that when the cellulose
material obtained from corn fiber is contacted at least once with
an alkaline bleaching agent after the alkaline material contacting
step, the glucose content of the cellulose material may be
increased. In a particularly preferred embodiment, the bleaching
agent comprises alkaline H.sub.2O.sub.2.
[0107] Further, it has been found to be critical that, when an
alkaline H.sub.2O.sub.2 is utilized as the bleaching agent, the
H.sub.2O.sub.2 must be applied in a step separate from the alkaline
extractant contacting step. Specifically, any alkaline
H.sub.2O.sub.2 treatment must be conducted after the alkaline
extraction contacting step and after the liquid comprising
arabinoxylan has been separated from the cellulose material. It has
been found that subjecting the corn fiber to an alkaline
H.sub.2O.sub.2 step without first conducting an alkaline extraction
contacting step and separating the alkaline extractant, such as in
the methods disclosed in WO 98/40413 (Doner et al.), results in
lower molecular weight arabinoxylan polymers being obtained.
Moreover, extraction of the corn fiber in this manner is very
dangerous to practice on an industrial scale because the heating of
alkaline H.sub.2O.sub.2 causes vigorous frothing and foaming and,
thus, may cause severe injuries and damage if extreme caution is
not practiced. The methods of the present invention do not cause
such hazards and are therefore more practicable on an industrial
scale. Accordingly, in a particularly preferred embodiment of the
invention herein, it is critical to separate the liquid comprising
arabinoxylan from the cellulose material prior to contacting the
cellulose material with the alkaline H.sub.2O.sub.2 bleaching
agent.
[0108] Further, as set forth in the Examples herein, if the corn
fiber is not subjected to an alkaline extractant contacting step of
greater than about 60.degree. C. prior to the alkali H.sub.2O.sub.2
step, reaction grade cellulose is not obtained. Therefore, it is
believed that the methods of Doner et al. do not allow one to
obtain corn fiber suitable for derivatization.
[0109] In one embodiment, the alkaline extracted cellulose material
is contacted with the alkaline H.sub.2O.sub.2 bleaching agent for
from about 5 to about 120 minutes at a temperature of from about
25.degree. C. to about 60.degree. C. In a further preferred
embodiment, the concentration of the alkaline H.sub.2O.sub.2 is
from about 1 wt. % to about 50 wt. %, further preferably, from
about 20 wt. % to about 40 wt. %, and, even more preferably, from
about 25 wt. % to about 35 wt. %. The pH of the alkaline
H.sub.2O.sub.2 is preferably from about 12 to about 14, and the
temperature is preferably from about 25.degree. C. to about
100.degree. C. If extreme caution is practiced, the alkaline
bleaching step may be conducted at from about 60.degree. C. to
about 100.degree. C. or at from about 25.degree. C. to about
100.degree. C. or from about 60.degree. C. to about 80.degree.
C.
[0110] In a still further preferred embodiment, the concentration
of the alkaline H.sub.2O.sub.2 is from about 0.5 M to about 5.0 M
and the pH is greater than about 10. When expressed as molar
equivalents, the preferred amount of alkaline H.sub.2O.sub.2 is
from about 0.1 to about 20 equivalents, with about 1 to about 4
equivalents being particularly preferred. In a further preferred
embodiment, when lower purity cellulose material i.e., between
about 50% to about 80% glucose is obtained from one or more
alkaline extractant contacting steps, it is possible to further
increase the purity of the cellulose material to greater than about
80% by treating this cellulose material with alkaline
H.sub.2O.sub.2.
[0111] In a further preferred embodiment, the cellulose material is
subjected to at least one alkaline H.sub.2O.sub.2 treatment. In yet
a further preferred embodiment, the cellulose material is subjected
to more than one alkaline H.sub.2O.sub.2 treatment. When the
cellulose material is subjected to one or more alkaline
H.sub.2O.sub.2treatments, it is critical to first separate the
liquid comprising arabinoxylani from the cellulose material before
conducting the first H.sub.2O.sub.2 bleaching.
[0112] Yet still further, the alkaline extracted cellulose material
from step (f) may be contacted at least one more time with an
alkaline extractant and the cellulose material so treated may be
treated with alkaline H.sub.2O.sub.2 at a concentration of from
about 0.5 M to about 4.0 M for about 5 to about 120 minutes,
thereby providing a cellulose material suitable for derivatization.
Again, it is necessary to separate at least the first alkaline
extractant from the cellulose fiber prior to conducting the
alkaline H.sub.2O.sub.2 treatment. Still further, the cellulose
material is contacted with the alkaline extractant at least two
additional times, thereby providing at least one additional liquid
comprising arabinoxylan. In a further preferred embodiment, at
least one additional alkaline extractant is added to the first
liquid comprising arabinoxylan. In a further preferred embodiment,
the cellulose material is contacted with the alkaline extractant
from about 2 to about 100 additional times, preferably in a
continuous method.
[0113] In yet a further preferred method of increasing the purity
of the cellulose material obtained from corn fiber, the cellulose
material is contacted with a xylanase enzyme after the alkaline
material contacting step for a time and at a temperature suitable
to increase the purity of the cellulose material. In a preferred
method, the xylanase enzyme contacting step is conducted at a
temperature of from about 25.degree. C. to about 100.degree. C. for
from about 0.1 h to about 24 h. Still further, the xylanase enzyme
contacting step is conducted at a temperature of from about
50.degree. C. to about 80.degree. C. for from about 1 h to about 4
h at a pH of from about 4 to about 9. In accordance with the
methods herein, in one embodiment, it has been found preferable to
subject the alkaline extractant and xylanase treated material to
one or more subsequent additional alkaline extractant contacting
steps, along with one or more optional alkaline H.sub.2O.sub.2
bleaching steps.
[0114] The xylanase enzymes preferred for use in this invention are
substantially free of cellulase activity. The xylanase enzymes that
fall within this description are available from a number of sources
(such as Genencor, Palo Alto, Calif.) and exhibit a wide range of
activity and conditions under which they exhibit maximum activity.
Again, one of ordinary skill in the art will recognize that the
exact conditions utilized for this and the other enzymatic
treatments herein will depend on the characteristics of the
particular enzyme utilized.
[0115] In a further preferred method of increasing the purity of
the cellulose material obtained from corn fiber, after treating the
corn fiber at least once with an alkaline extractant, the alkaline
extracted corn fiber is subjected to at least one acid rinsing
step. In one embodiment, the acid is an organic acid. In a further
embodiment, the acid in the acid rinse is in an amount of from
about 10 wt. % to about 95 wt. % acetic acid. Still further, the
acid in the acid rinsing step may be an inorganic acid. In yet
another embodiment, the acid in the acid rinsing step is in an
amount of from about 10 wt. % to about 20 wt. % of a sulfuric acid
solution. In a further embodiment, the acid rinsing step is
conducted at from about 60.degree. C. to about 100.degree. C. The
acid rinsing treatment may optionally be followed by an alkaline
bleaching step. Thus, in a further embodiment, the acid rinsing
step is added between the alkaline extractant contacting step and
prior to the at least one treatment with alkaline
H.sub.2O.sub.2.
[0116] In a further major aspect, the invention provides a method
of making cellulose derivatives from corn fiber. In one embodiment,
the cellulose material has been subjected to at least one alkaline
extractant contacting step. In a further embodiment, the corn fiber
cellulose utilized to prepare the cellulose derivatives has been
subjected to at least one alkaline material contacting step,
followed by at least one alkaline H.sub.2O.sub.2 bleaching step.
Still further, it is preferred that the cellulose utilized to
prepare the cellulose derivatives has been subjected to at least
one alkaline extractant step, followed by at least one acid
contacting step followed by at least one alkaline H.sub.2O.sub.2
bleaching step. Yet still further, it is preferred that the
cellulose material has been subjected to at least one alkaline
extractant contacting step, followed by at least one xylanase
enzyme contacting step, then followed by at least one alkaline
H.sub.2O.sub.2 bleaching step. In a particularly preferred
embodiment, the cellulose material utilized to prepare the
cellulose derivatives has been subjected to at least two alkaline
material steps, followed by at least one alkaline H.sub.2O.sub.2
bleaching step. In separate, still preferred embodiments, the
cellulose material may be treated in any order with one or more
alkaline H.sub.2O.sub.2 contacting steps, one or more acid
contacting steps and/or one or more xylanase enzyme contacting
steps, provided that the cellulose material is obtained from
proteolyzed corn fiber subjected to at least one alkaline
extractant contacting step, and when the subsequent treatment is
with alkaline H.sub.2O.sub.2, the alkaline extractant is separated
from the cellulose material prior to the performing of any
additional steps.
[0117] In one embodiment, the invention provides a method of making
cellulose esters from corn fiber. In a further embodiment, the
invention provides a method of making cellulose ethers from corn
fiber.
[0118] In one embodiment of the method of obtaining cellulose
esters from corn fiber, the method comprises contacting the
cellulose material obtained according to the methods described
above with a C1 to C10 esterifying agent, preferably in the
presence of a catalyst, thereby providing a cellulose ester. Prior
to performing the esterification reaction, the cellulose material
may optionally be subjected to at least one additional alkaline
extractant contacting step, an alkaline H.sub.2O.sub.2 bleaching
step, a xylanase enzyme contacting step, or a mixture of any of
these steps in any combination, provided that at least one alkaline
extractant contacting step is conducted first and the first liquid
comprising arabinoxylan is removed from the cellulose material
prior to the conducting of any subsequent alkaline H.sub.2O.sub.2
bleaching step. A hot water (>60.degree. C.) rinsing step may
optionally be conducted after the first alkaline extraction step
and/or between any subsequent treatment steps.
[0119] In a preferred embodiment, the esterifying agent comprises a
C1 to C10 acyl anhydride, acyl acid, acyl halide, or a mixture
thereof. Still further, it is preferable that the esterifying agent
comprises formic anhydride, acetic anhydride, propionic anhydride,
butyric anhydride, or a mixture thereof. Yet still further, it is
preferable that the esterifying agent comprises acetic anhydride,
propionic anhydride or butyric anhydride.
[0120] In one embodiment, the cellulose esters are prepared
utilizing H.sub.2SO.sub.4 as a catalyst. In a further embodiment,
the cellulose esters are prepared utilizing TFAA (trifluoroacetic
acid) or MSA (methane sulfonic acid), or a mixture thereof as a
catalyst.
[0121] In one embodiment, it is particularly preferable that the
cellulose esters be prepared from corn fiber cellulose material
that has been subjected to at least one alkaline extractant
contacting step, followed by bleaching with alkaline
H.sub.2O.sub.2. With use of the high purity cellulose obtained, it
is possible to prepare high molecular weight cellulose esters.
Generally, it is believed that higher molecular weight cellulose
esters may be obtained when TFAA or MSA is utilized because this
material degrades the cellulose material less than does
H.sub.2SO.sub.4. Further, TFAA and MSA are believed to be more
effective in esterifying non-glucose monomers and solubilizing any
low molecular weight polymers present in the cellulose obtained
from corn fiber.
[0122] As noted, in the preferred methods of the invention, the
cellulose is preferably maintained as about 80% or greater in
cellulose I. Therefore, it is not necessary to conduct a classical
activation of the cellulose in order to prepare the derivatives
herein. One of ordinary skill in the art will recognize specific
methods to prepare cellulose esters; accordingly, such methods are
not described in detail herein. Methods to prepare cellulose esters
that are suitable for use herein are described generally in U.S.
Pat. Nos. 1,698,049, 1,683,347, 1,880,808, 1,880,560, 1,984,147,
2,129,052, and 3,617,201; the disclosures of each of these patents
are incorporated herein by this reference in their entireties.
[0123] In a further embodiment of the invention herein, the
cellulose esters obtained from corn fiber comprise cellulose
formate, cellulose acetate, cellulose propionate, cellulose
butyrate, cellulose formate acetate, cellulose formate propionate,
cellulose formate butyrate, cellulose acetate propionate, cellulose
acetate butyrate, or a mixture thereof. Yet still further, the
cellulose esters comprise cellulose acetate, cellulose acetate
propionate or cellulose acetate butyrate, or a mixture thereof. It
is preferred that the cellulose esters obtained according to the
methods herein have DS/AGU's of from about 0.1 to about 3.0. Still
further, it is preferred that the cellulose esters obtained
according to the methods herein have DS/AGU's of from about 1.7 to
about 2.75. Still further, it is preferable that the cellulose
esters have inherent viscosities of from about 0.001 to about
3.0.
[0124] In a further embodiment, it is preferred that prior to
esterification, the cellulose material be subjected to at least one
additional alkaline extractant step, followed by at least one
treatment with alkaline H.sub.2O.sub.2. If an alkaline
H.sub.2O.sub.2 bleaching step is utilized, the H.sub.2O.sub.2 is
preferably at a concentration of from about 0.5 to about 4.0 M for
about 5 to about 120 minutes. Further preferably, prior to
esterification, the cellulose material is contacted with an acid
rinse after the alkaline extractant contacting step. Still further,
prior to esterification, the alkaline extracted cellulose material
is treated with a xylanase enzyme.
[0125] In one embodiment of the method of obtaining cellulose
ethers from corn fiber, the method comprises contacting the
cellulose material obtained from the methods described above with
an O-alkylating agent, thereby providing a cellulose ether. Prior
to performing the etherification reaction, the cellulose material
may optionally be subjected to at least one additional alkaline
extractant contacting step, an alkaline H.sub.2O.sub.2 bleaching
step, a xylanase enzyme contacting step, or a mixture of any of
these steps in any combination, provided that at least one alkaline
extractant contacting step is conducted first and the liquid
comprising arabinoxylan is removed from the cellulose material
prior to the conducting of any subsequent alkaline H.sub.2O.sub.2
bleaching step. A hot water (>60.degree. C.) rinsing step may
optionally be conducted after the first alkaline extraction step
and/or between any subsequent treatment steps.
[0126] In a preferred embodiment of this method, the O-alkylating
agent comprises alkylene oxide, aryl substituted alkylene oxide,
halogen substituted alkylene oxide, alkyl halide, hydroxyalkyl
halide, aryl alkyl halide, carboxyalkyl halide,
(alkyloxycarbonyl)alkyl halide, allyl halide, vinyl halide, alkyl
sulfonate, hydroxyalkyl sulfonate, aryl alkyl sulfonate,
carboxyalkyl sulfonate, (alkyloxycarbonyl)alkyl sulfonate, allyl
sulfonate, or a mixture thereof. Still further preferably, the
O-alkylating agent comprises ethylene oxide, propylene oxide,
butylene oxide, epoxybutene, amylene oxide, glycidol, styrene
oxide, epichlorohydrin, methyl chloride, methyl iodide, methyl
bromide, ethyl bromide, propyl bromide, butyl bromide, propyl
methyl sulfonate, methyl chloroacetic acid, ethyl chloroacetic
acid, sodium chloroacetate, chloroacetic acid, benzyl bromide,
1-N,N-dialkylamino-2-chloroethane, or a mixture thereof. Yet still
further, the O-alkylating agent comprises ethylene oxide, propylene
oxide, epoxybutene, methyl chloride, ethyl bromide, sodium
chloroacetate, or a mixture thereof. In a particularly preferred
embodiment, the O-alkylating agent is epoxybutene.
[0127] In further preferred embodiments, the cellulose ethers
preferred according to the methods herein have a DS/AGU of from
about 0.01 to about 3.0. In a still further preferred embodiment of
the invention herein, the cellulose ethers have a DS/AGU of from
about 0.3 to about 2.2. Yet still further, the cellulose ethers
prepared according to the methods herein have MS/AGU's of from
about 0.01 to about 100. More preferably, the cellulose ethers
prepared according to the methods herein have MS/AGU's of from
about 0.1 to about 5.
[0128] In a further embodiment, it is preferred that prior to
etherification, the cellulose material be subjected to at least one
additional alkaline extractant step, followed by at least one
treatment with alkaline H.sub.2O.sub.2. If an alkaline
H.sub.2O.sub.2 treatment is provided, the treatment is preferably
conducted at a concentration of from about 0.5 to about 4.0 M at a
time of from about 5 to about 120 minutes. Further preferably,
prior to etherification, the cellulose material is contacted with
an acid rinse after the alkaline extractant contacting step. Still
further, prior to etherification, the alkaline extracted cellulose
material is treated with a xylanase enzyme.
[0129] In a further major embodiment, the invention relates to a
method of extracting arabinoxylan from corn fiber wherein the
method comprises the steps of: (a) heating an aqueous mixture of
corn fiber and a liquid; (b) contacting the mixture of step (a)
with a protease enzyme, thereby providing a proteolyzed corn fiber
and a liquid; (c) separating the liquid from the proteolyzed corn
fiber; (d) contacting the proteolyzed corn fiber at least once with
an alkaline extractant, thereby providing an insoluble cellulose
material and a liquid comprising arabinoxylan; (e) separating the
insoluble cellulose material from the liquid comprising
arabinoxylan, and (f) reducing the volume of the liquid comprising
arabinoxylan by removing excess alkaline extractant, thereby
providing a concentrated liquid wherein the liquid comprises from
about 10% to about 50% solids and wherein the solids comprise
arabinoxylan. In a further embodiment, the alkali utilized in the
alkaline extractant contacting step is not alkaline
H.sub.2O.sub.2.
[0130] The proteolyzed corn fiber utilized in this aspect of the
invention may be destarched and/or solvent extracted as disclosed
above. In one embodiment, the alkaline extractant comprises the
alkaline material utilized in the methods disclosed above. Further,
the corn fiber cellulose may be subjected to one or more alkaline
H.sub.2O.sub.2 bleaching, acid rinses and/or xylanase enzyme
treatments also as disclosed above.
[0131] In a particularly preferred embodiment, in conjunction with
the alkaline extraction step, the insoluble cellulose material is
separated from the liquid comprising arabinoxylan at a temperature
of at or above about 60.degree. C. In accordance with the methods
herein, it has surprisingly been found that by maintaining the
temperature of the liquid comprising arabinoxylan at or above about
60.degree. C., the viscosity of the liquid comprising arabinoxylan
(and the concentrated liquid) may be lowered substantially. This
lower viscosity significantly increases the processability of the
liquid comprising arabinoxylan (and the concentrated liquid).
Further, as discussed in more detail above, if the alkali extracted
cellulose material is to be utilized for derivatization, it is also
particularly preferred that the cellulose material be separated
from the alkali extractant comprising arabinoxylan at a temperature
of at or above about 60.degree. C.
[0132] Further, according to the methods herein, it has
surprisingly been found that extraction of the arabinoxylan from
unground corn fiber can be accomplished in less than about 2 h,
more preferably, less than 1 h. It is believed that such results
result because temperatures of above 60.degree. C. are utilized
herein. Significantly, such elevated temperatures are not generally
possible with methods that utilize alkaline H.sub.2O.sub.2 to
extract arabinoxylan because of the hazards associated with heating
alkaline H.sub.2O.sub.2 to over 60.degree. C. Thus, prior art
methods incorporating H.sub.2O.sub.2 to extract arabinoxylan
generally utilize lower temperatures and, as such, require longer
processing times to suitably extract cellulose from corn fiber.
Further, with the methods herein, extraction of arabinoxylan at
temperatures greater than about 60.degree. C. at times less than 2
hours provided yields of arabinoxylan greater than about 25 wt. %,
as measured by dry weight of corn fiber. Moreover, it has also been
found that the arabinoxylan obtained according to the methods
herein is not off-white, but is essentially white, with little or
no "off color."
[0133] In a further preferred embodiment, the liquid comprising
arabinoxylan is subjected to a vacuum distillation step
concurrently with the volume reducing step. In accordance with the
methods herein, prior to precipitation of the arabinoxylan, it has
been found to be critical in some circumstances to concentrate the
liquid comprising arabinoxylan to from about 10 wt. % to about 80
wt. % solids and, more preferably, from about 40 wt. % to about 60
wt. % solids. In a particularly preferred embodiment, the solids
level of the concentrated liquid is from about 10 wt. % to about 50
wt. %. Still further, the solids level is greater than about 15 wt.
%. In a further embodiment, the solids level is from about 15 wt. %
to about 50 wt. %. In a further, still preferred embodiment, the
solids level of the concentrated liquid is from about 20 wt. % to
about 50 wt. %.
[0134] In one particularly preferred embodiment, the liquid
comprising arabinoxylan is concentrated by heating the liquid under
vacuum while providing an airflow to the surface of the solution
being concentrated. By utilizing this method, foaming of the
solution can be minimized. In another preferred embodiment, the pH
of the liquid comprising arabinoxylan can be adjusted to from about
4.5 to about 5.0 before concentration of the liquid comprising
arabinoxylan. By this method, foaming of the solution can also be
minimized or eliminated.
[0135] The volume reduction of the liquid comprising arabinoxylan
may be conducted in any number of ways known to one of ordinary
skill in the art, such as by simple distillation or by steam
purging while continuously conveying the extracted arabinoxylan
solution. In a particularly preferred embodiment, the volume
reducing step may be conducted via ultrafiltration. It is preferred
that the alkaline extractant be removed from the liquid comprising
arabinoxylan concurrently with an ultrafiltration volume reducing
step. In this method, the liquid comprising arabinoxylan is
conveyed through an arabinoxylan-rejecting membrane. The
arabinoxylan will thus remain on one side of the membrane, while at
least some liquid comprising alkaline material will be conveyed
through the membrane. As a result, the liquid comprising
arabinoxylan will become increasingly more concentrated as the
process proceeds, thereby providing the concentrated liquid. One of
ordinary skill in the art will recognize the methods that may be
utilized to select the appropriate membrane and to perform the
ultrafiltration methods.
[0136] In a further preferred embodiment, a precipitation agent is
added to the concentrated liquid, thereby providing a precipitate
consisting essentially of arabinoxylan. In a particularly preferred
embodiment, the precipitation agent comprises acetic acid or
propionic acid. Even more preferably, the precipitation agent is
acetic acid. Still further preferably, the separating and volume
reducing steps are each conducted at a temperature of at or above
about 60.degree. C., the volume reducing step takes place at a pH
of above about 4.5 and the precipitation agent comprises acetic
acid.
[0137] In a preferred embodiment, the precipitation agent comprises
from about 0.1 to about 6.0 volumes of acetic acid based upon the
weight of arabinoxylan in the concentrated liquid and wherein the
acetic acid and the concentrated liquid are each at a temperature
of from about 0.degree. C. to about 60.degree. C. or, still
preferably, from about 20.degree. C. to about 30.degree. C. when
the acetic acid is added to the concentrated liquid. Still further
preferably, the precipitation agent comprises from about 1.0 to
about 3.0 volumes of acetic acid based upon the weight of
arabinoxylan in the concentrated liquid and wherein the acetic acid
and the concentrated liquid are each at a temperature of from about
0.degree. C. to about 60.degree. C., or still preferably, are each
at a temperature of from about 20.degree. C. to about 30.degree.
C., when the acetic acid is added to the concentrated liquid. Yet
further preferably, the temperature of the precipitation agent and
the concentrated liquid are nearly the same at the time of adding
the acetic acid.
[0138] It has been surprisingly found that when acetic acid is
utilized as the precipitation agent, arabinoxylan of an especially
white color (or comprising a low yellow index) can be obtained.
Since it is especially desirable to obtain a white arabinoxylan
precipitate for certain applications, in a particularly preferred
example, acetic acid is utilized as the precipitation agent,
thereby providing an essentially white powder. One of ordinary
skill in the art will recognize the methods utilized to measure
whiteness (or lack of color). It has been determined that with the
methods of the present invention it is possible to prepare an
arabinoxylan that is suitable for applications requiring a white
powder or applications involving solutions of the arabinoxylan
having little color. It has also been found that it is possible to
prepare low color arabinoxylan solutions that have very little
taste, a quality which is beneficial for food and pharmaceutical
applications.
[0139] In separate embodiments, the acetic acid may be added to the
concentrated liquid in at least two ways. First, the acetic acid
can be added cold. Addition of cold acetic acid results in a rapid
precipitation of the arabinoxylan. While this rapid precipitation
is believed to cause the arabinoxylan to have a higher salt
content, for those applications where arabinoxylan salt content is
not relevant, this method may be preferred. In the second, more
preferred, method of acetic acid addition, room temperature acetic
acid is added to the concentrated liquid, wherein the concentrated
liquid may be at a temperature of at or below about 60.degree. C.
This results in a slower precipitation of arabinoxylan which
results in a markedly lower salt content in the arabinoxylan.
Preferably, the temperatures of the precipitation agent and the
concentrated liquid are nearly the same at the time of
precipitation and the temperature of both components is from about
20.degree. C. to about 30.degree. C. It is particularly preferred
that the temperature of both the concentrated liquid and the acetic
acid are each at a temperature of from 0.degree. C. to below about
60.degree. C. Still further preferably, the temperature of both the
concentrated liquid and the acetic acid are each at a temperature
of from 20.degree. C. to about 40.degree. C.
[0140] Further, with the invention herein, it has been found that
when acetic acid is added slowly and, consequently, the pH of the
concentrated liquid is lowered more slowly, it is possible to
separate out two components from the concentrated liquid. The first
(and minor) component is hemicellulose A. This hemicellulose A
material precipitates at approximately pH 4.5. As used herein, the
term "hemicellulose A" means that portion of corn fiber
arabinoxylan that, once precipitated and dried, is not soluble in
water at acidic or neutral pH. Specifically, in order to redissolve
the hemicellulose A, the pH must be adjusted to at least pH 12 to
13. Furthermore, hemicellulose A is characterized by having a high
glucose content (greater than about 20%) and a low molecular weight
(less than about 20,000). The second (and major) component that
precipitates from the concentrated liquid is hemicellulose B.
Hemicellulose B contains little glucose (from about 2% or less) and
is fully water soluble (even after drying) in water at a full range
of pH's i.e., from acid to alkaline.
[0141] In one embodiment, hemicellulose A is first precipitated and
removed from the concentrated liquid during the precipitation step,
thereby providing a concentrated liquid, wherein the arabinoxylan
consists essentially of hemicellulose B. In yet a further
embodiment, the hemicellulose B is precipitated from the
concentrated liquid, thereby providing an arabinoxylan precipitate
consisting essentially of hemicellulose B. In a further preferred
embodiment, the average weight molecular weight of the arabinoxylan
is greater than 300,000.
[0142] As discussed further below, the type of arabinoxylan
utilized affects the preparation of arabinoxylan derivatives.
Therefore, in one particularly preferred example, hemicellulose A
is separated from the concentrated liquid during a precipitation
step, thereby providing an arabinoxylan consisting essentially of
hemicellulose B. In a further preferred example, the arabinoxylan
precipitate comprises less than about 8% of hemicellulose A. Still
further, hemicellulose A may first be precipitated and removed from
the concentrated liquid thereby providing a second concentrated
liquid, wherein the second concentrated liquid consists essentially
of hemicellulose B. It is further preferred that the hemicellulose
B be separated from the second concentrated liquid.
[0143] In yet a further preferred embodiment, the precipitation
agent comprises acetic acid at a concentration of from about 0.1
wt. % to about 25 wt. %, based upon the weight of arabinoxylan in
the concentrated liquid, wherein the acetic acid and the
concentrated liquid are each at a temperature of from about
20.degree. C. to about 60.degree. C., or from about 20.degree. C.
to about 35.degree. C., thereby providing an arabinoxylan
precipitate with an inorganic salt content of less than about 5.0%
as measured by dry weight of the arabinoxylan. In a still further
preferred embodiment, the precipitation agent comprises acetic acid
at a concentration of from about 1.0 to about 6.0 volumes or from
about 1.0 to about 3.0 volumes of acetic acid, based upon the
weight of arabinoxylan in the concentrated liquid, wherein the
acetic acid and the concentrated liquid are each at a temperature
of from about 20.degree. C. to about 60.degree. C., or from about
20.degree. C. to about 35.degree. C., thereby providing an
arabinoxylan precipitate with an inorganic salt content of less
than about 5.0% as measured by dry weight of the arabinoxylan. The
amount of salt in the arabinoxylan may also be reduced according to
the preferred ultrafiltration concentration method discussed
above.
[0144] Use of acetic acid as a precipitation agent provides the
added benefit of reducing foaming of the liquid comprising
arabinoxylan that may occur during the volume reducing step. The
organic salt that forms, such as sodium acetate, is not isolated
with the precipitated arabinoxylan to any significant extent. In an
alternate embodiment, when the amount of salt in the arabinoxylan
is not important, foaming of the liquid comprising arabinoxylan may
be minimized by the addition of an inorganic acid, such as HCl or
H.sub.2SO.sub.4. Acidification of the liquid comprising
arabinoxylan with these acids leads to the formation of inorganic
salts, which are not generally removed from the arabinoxylan during
precipitation or washing. Thus, addition of inorganic acids is
normally not preferred unless salt content of the arabinoxylan is
unimportant.
[0145] In a further embodiment, with respect to the concentrated
liquid, hemicellulose A may be separated from hemicellulose B
concurrently with the volume reducing step by utilizing
ultrafiltration. One of ordinary skill in the art will recognize
that a specifically selective membrane may be utilized which allows
the lower molecular weight hemicellulose A to pass through the
membrane, but does not allow the higher molecular weight
hemicellulose B to flow through the membrane. As a result of this
ultrafiltration separation method, it is possible to cleanly
separate hemicellulose B with minimal concern about inorganic salt
content. In a particularly preferred embodiment, the alkaline
extractant is removed from the liquid comprising arabinoxylan
concurrently with the ultrafiltration volume reducing step. The
hemicellulose A and B obtained according to this ultrafiltration
separation method are preferably identical in characteristics to
the hemicellulose obtained according to the precipitation methods
discussed herein. However, it is noted that when the
ultrafiltration hemicellulose separation method is utilized, any
inorganic salts in the separated hemicellulose may be maintained at
significantly less than about 5.0 wt. %. This allows the at least
three or less molecular weight fraction materials to be separated
from the concentrated liquid without performing a separate
precipitation step. In a further embodiment, the arabinoxylan may
be separated directly from the liquid comprising arabinoxylan via
ultrafiltration as a mixture of both hemicellulose A and B without
first needing to perform a volume reducing step.
[0146] In the practice of this invention, it is found that the
arabinoxylan obtained by the methods disclosed herein is different
in structure, molecular weight, and physical properties than the
hemicelluloses disclosed in the prior art, for example, in Glasser
et al. (U.S. Pat. No. 5,430,142). Characterization of the
hemicelluloses of Glasser et al. have been reported by Glasser
(U.S. Pat. No. 5,430,142) and by Gabrielii ("Hydrogels based on
hardwood hemicelluloses" Licentiate thesis, Chalmers University of
Technology, Goteborg, Sweden. ISBN 91-7197-697-3).
[0147] The hemicellulose of Glasser is a xylan comprised almost
exclusively of xylose with small amounts of uronic acid side chains
(approximately 1 per 8 xylose residues). The molecular weight was
reported to be less than 100,000, as determined according to the
methods in the Glasser reference. Furthermore, the hemicellulose of
Glasser was reported to be soluble only in aqueous alkali of a pH
greater than approximately 10. In contrast, as is set out herein,
the arabinoxylan of the present invention is substantially
branched, in which the branches are comprised of xylose, arabinose,
galactose, glucouronic acid, and 4-O-methyl glucouronic acid or a
mixture thereof. Further, the arabinoxylan of the present invention
has a weight-average molecular weight of greater than about 300,000
and is soluble in water at a pH of from about 1 to about 14.
[0148] As for the differences, analyses indicate that the corn
fiber-derived arabinoxylan obtained according to the methods herein
is different from the hemicellulose obtained according to the
methods disclosed in the prior art, for example, the method of
Glasser et al. measured according to the methods herein (U.S. Pat.
No. 5,430,142). That is, analyses performed according to the
invention herein indicate that the hemicellulose of Glasser et al.
is comprised of at least 4 to 5 molecular weight fractions, as
measured by gel permeation chromatography. Further, analyses have
shown that the arabinoxylan of Glasser et al. has a molecular
weight of less than about 250,000 as measured according to the
methods herein. Accordingly, the arabinoxylan obtained from corn
fiber according to the methods herein has a much higher molecular
weight than the hemicellulose disclosed in the prior art. Further,
it is believed that the extraction techniques utilized to obtain
the arabinoxylan will significantly affect the molecular weight of
the arabinoxylan obtained. Accordingly, it is believed that the
arabinoxylan obtained by the methods herein has not before been
extracted. As such, products made by the processes herein are
believed to be novel.
[0149] In a further embodiment, arabinoxylan in the liquid
comprising arabinoxylan and the concentrated liquid consists
essentially of at least two but less than four molecular weight
fractions as measured by gel permeation chromatography. Still
further, the arabinoxylan in the liquid comprising arabinoxylan and
the concentrated liquid consists essentially of exactly three
molecular weight fractions as measured by gel permeation
chromotography.
[0150] Based upon the above, it is preferred that at least about
92% of the arabinoxylan in the concentrated liquid, as well as that
in the liquid comprising arabinoxylan, has a weight-average
molecular weight of from about 300,000 to about 2,000,000. In a
further preferred embodiment, at least about 92% of the
arabinoxylan in the concentrated liquid and the liquid comprising
arabinoxylan has a weight-average molecular weight of from about
400,000 to about 700,000. In a further preferred embodiment, at
least about 92% of the arabinoxylan in the concentrated liquid and
the liquid comprising arabinoxylan has a weight-average molecular
weight of greater than about 300,000. In an alternate, still
preferred embodiment, the arabinoxylan separated from the
concentrated liquid and the liquid comprising arabinoxylan, whether
by precipitation, by ultrafiltration or some other method, consists
essentially of hemicellulose B and this hemicellulose B has a
weight average molecular weight of greater than about 300,000. In a
particularly preferred embodiment, the arabinoxylan separated from
the concentrated liquid (or directly separated from the liquid
comprising arabinoxylan) comprises less than about 8% of
hemicellulose A.
[0151] In a further embodiment, it is preferred that the
arabinoxylan in the concentrated liquid and the liquid comprising
arabinoxylan is comprised of a xylan main polymer chain with at
least two branches, wherein the branches comprise groups of xylose,
arabinose, galactose, glucouronic acid, 4-O-methyl glucouronic
acid, or a mixture thereof. Still further, it is preferred that the
arabinoxylan obtained according to the methods herein is soluble in
water at a pH of from about 1 to about 14. In yet a further
preferred embodiment, the arabinoxylan comprises a branched
polysaccharide having a weight-average molecular weight of greater
than about 300,000. In still a further embodiment, the arabinoxylan
in the liquid comprising arabinoxylan and in the concentrated
liquid comprising arabinoxylan comprises a highly branched
polysaccharide, essentially soluble in water from a pH of from
about 1 to about 10, and having a weight-average molecular weight
greater than about 300,000.
[0152] It is further preferred that the rate of arabinoxylan
extraction from the corn fiber during the alkaline material
contacting step is measured in situ via infrared spectroscopy.
[0153] In accordance with the methods herein, it is possible to
react the arabinoxylan obtained from corn fiber in order to prepare
arabinoxylan derivatives. Specifically, it is possible to prepare
novel arabinoxylan esters and ethers.
[0154] With respect to the arabinoxylan esters, in a preferred
embodiment, the invention comprises contacting the separated
arabinoxylan in a reaction medium with a C1 to C10 esterifying
agent in the presence of a catalyst, thereby providing an
arabinoxylan ester. In a further embodiment, the separated
arabinoxylan utilized for preparing the arabinoxylan esters herein
consists essentially of hemicellulose B. In this embodiment,
hemicellulose B may be separated from hemicellulose A according to
the methods set out above. In yet a further embodiment, the
arabinoxylan utilized to prepare the arabinoxylan ester comprises a
mixture of hemicellulose A and hemicellulose B.
[0155] In accordance with the methods herein, it has been found
possible to prepare the arabinoxylan esters from an arabinoxylan
precipitate without first drying the separated arabinoxylan
precipitate. Thus, in one particularly preferred embodiment, the
arabinoxylan is separated from the concentrated liquid via
precipitation with acetic acid and the separated arabinoxylan is
wet with acetic acid prior to contacting with the esterifying
agent. In some circumstances, it has been found that the ability to
form esters from the arabinoxylan is decreased if the precipitated
arabinoxylan is dried to remove acetic acid prior to
derivatization. Thus, in one particularly preferred embodiment, it
is critical that the arabinoxylan be kept wet with acetic acid
prior to treatment with the esterifying agent. In other words, when
preparing the arabinoxylan esters, the acetic acid should not be
removed from the separated arabinoxylan, such as by rinsing. A
surprising benefit seen with this embodiment is that the rate of
formation of the arabinoxylan ester is faster and the molecular
weight of the arabinoxylan ester is higher relative to when the
arabinoxylan is dried prior to contacting the arabinoxylan with the
esterifying agent.
[0156] In a further embodiment, the arabinoxylan may be separated
from the concentrated liquid or the liquid comprising arabinoxylan
via ultrafiltration according to the methods discussed herein. When
separation is in accordance with this method, acetic acid may be
added to the separated arabinoxylan prior to contacting the
arabinoxylan with the esterifying agent in order to obtain the
benefits seen with performing an esterification reaction with a
precipitated arabinoxylan that is wet with acetic acid.
[0157] In yet a further embodiment relating to the arabinoxylan
esters, the catalyst comprises trifluoroacetic anhydride,
trifluoromethane sulfonic acid, C1-C12 alkyl sulfonic acid, C1-C12
aryl sulfonic acid, C1-C12 substituted aryl sulfonic acid, or a
mixture thereof. Still further, the catalyst preferably comprises
trifluoroacetic anhydride, methane sulfonic acid, p-toluene
sulfonic acid, or a mixture thereof. In a particularly preferred
embodiment, the catalyst comprises methane sulfonic acid.
[0158] The catalyst may preferably be present at from about 0.0001
to about 100 equivalents per hydroxyl, more preferably from about
0.01 to about 10 equivalents per hydroxyl. The preferred reaction
temperatures are from about 0.degree. C. to about 100.degree. C.,
preferably from about 25.degree. C. to about 70.degree. C., and,
most preferably, at equal to or less than about 60.degree. C. It is
preferred that the esterifying agent contacting step is conducted
for from about 0.1 h to 100 h, more preferably from about 1 h to
about 100 h, and even more preferably from about 1 h to about 20 h.
In yet a further embodiment, the reaction time is from about 1 h to
about 2 h and the reaction temperature is from about 25.degree. C.
to about 50.degree. C. Still preferably, the reaction time is from
about 1 h to about 4 h and the temperature is less than about
60.degree. C.
[0159] In a further embodiment, the catalyst utilized to prepare
the arabinoxylan esters is a Lewis acid. Still further, the
reaction mixture utilized to prepare the arabinoxylan esters
comprises an organic solvent, wherein the organic solvent is not an
acyl acid or acyl anhydride. In yet a further preferred embodiment,
the reaction mixture comprises an organic solvent and wherein the
organic solvent is DMAC or DMF.
[0160] In still a further embodiment, the reaction time when the
catalyst is Lewis acid is from about 0.1 h to about 100 h, more
preferably the reaction time is from about 1 to about 20 h. In a
separate embodiment, the reaction mixture is at a temperature of
from about 0.degree. C. to about 180.degree. C. The amount of Lewis
acid catalyst may range from about 0.0001 to about 100 equivalents
per hydroxyl, more preferably from about 0.01 to about 10
equivalents per hydroxyl. In a preferred embodiment, the catalyst
comprises TiCl.sub.4, Ti(O.sup.iPr).sub.4, or SnCl.sub.4, the
esterifying agent contacting time is from about 1 to about 20 h,
and the contacting temperature is from about 50.degree. C. to about
150.degree. C.
[0161] Still in relation to the arabinoxylan esters, in a further
embodiment, the catalyst may be an inorganic acid. Inorganic acids
useful in the methods of this invention are H.sub.2SO.sub.4, HCl,
NaHSO.sub.4, HClO.sub.4, and H.sub.3PO.sub.4. In this embodiment,
the esterifying agent contacting time may be from about 0.1 h to
about 100 h and the temperature may be from about 20.degree. C. to
about 90.degree. C. The amount of catalyst may range from about
0.0001 to about 100 equivalents per hydroxyl, more preferably from
about 0.01 to about 10 equivalents per hydroxyl. More preferably,
the esterifying agent contacting step may be conducted for from
about 1 h to about 20 h, at a temperature of from about 25.degree.
C. to about 70.degree. C. or from about 40.degree. C. to about
70.degree. C.
[0162] In a particularly preferred embodiment of the arabinoxylan
ester preparation methods herein, the catalyst comprises
NaHSO.sub.4, the reaction time is from about 1 to about 20 h and
the reaction temperature is from about 25.degree. C. to about
70.degree. C.
[0163] With respect to the esterifying agents, such materials may
comprise a C1 to C10 acyl anhydride, C1-C10 acyl acid, C1-C10 acyl
halide, or a mixture thereof. Still further, the esterifying agent
may comprise formic anhydride, acetic anhydride, propionic
anhydride, butyric anhydride, or a mixture thereof.
[0164] As for the types of arabinoxylan esters prepared according
to the methods herein, the arabinoxylan ester comprises
arabinoxylan formate, arabinoxylan acetate, arabinoxylan
propionate, arabinoxylan butyrate, arabinoxylan formate acetate,
arabinoxylan formate propionate, arabinoxylan formate butyrate,
arabinoxylan acetate propionate, arabinoxylan acetate butyrate or a
mixture thereof. In a further embodiment, the arabinoxylan ester
comprises arabinoxylan formate, arabinoxylan propionate,
arabinoxylan butyrate, arabinoxylan formate acetate, arabinoxylan
formate propionate, arabinoxylan acetate propionate, arabinoxylan
acetate butyrate, or a mixture thereof. In one embodiment, the
arabinoxylan ester does not comprise arabinoxylan acetate.
[0165] In a further preferred embodiment, the arabinoxylan ester
consists essentially of an esterified hemicellulose B. Still
further preferred, the arabinoxylan ester has a DS/CU of from about
0.1 to about 2.5. Yet further preferred, the arabinoxylan ester has
a DS/CU of from about 2.0 to about 2.4 In a further embodiment, the
arabinoxylan ester has a weight-average molecular weight of greater
than about 50,000. In a particularly preferred embodiment, the
arabinoxylan ester has a DS/CU of from about 2.0 to about 2.4 and a
weight-average molecular weight of greater than about 150,000.
[0166] Novel arabinoxylan esters are also prepared according to the
methods herein. Such arabinoxylan esters are prepared as the
product of the reaction of the arabinoxylan obtained according to
the methods herein with a C1 to C10 esterifying agent wherein the
reaction is conducted in the presence of a suitable catalyst. In
one embodiment, the novel arabinoxylan ester is prepared from an
essentially branched and water soluble arabinoxylan consisting
essentially of an arabinoxylan of a weight average molecular weight
of greater than about 300,000. Still further, the novel
arabinoxylan esters of this invention are prepared from an
arabinoxylan comprised of a xylan main polymer chain with at least
two branches, wherein the branches comprise groups of xylose,
arabinose, galactose glucouronic acid, 4-O-methyl glucouronic acid,
or a mixture thereof. Yet still further, the novel arabinoxylan
esters are prepared from an arabinoxylan which is essentially
soluble in water at a pH of from about 1 to about 14.
[0167] Still further the invention provides arabinoxylan esters
prepared by the methods herein.
[0168] Arabinoxylan ethers are also preferably made according to
the invention herein. In one embodiment, the invention comprises,
in a reaction medium, contacting the concentrated liquid or the
liquid comprising arabinoxylan with an O-alkylating agent, thereby
providing an arabinoxylan ether. The pH of the reaction medium is
preferably greater than about 10. In one preferred embodiment, the
O-alkylating agent is added directly to the concentrated liquid or
the liquid comprising arabinoxylan. In yet a further embodiment, a
precipitation agent may be added to the concentrated liquid as set
out above, thereby forming an arabinoxylan precipitate. Further, as
discussed above, the arabinoxylan may be separated from the
concentrated liquid or the liquid comprising arabinoxylan via
ultrafiltration. This separated arabinoxylan may then be contacted
with an O-alkylating agent at a pH of preferably greater than about
10, thereby providing an arabinoxylan ether.
[0169] In one embodiment, the reaction medium comprises water to
provide an aqueous reaction medium. In yet a further embodiment,
the reaction medium comprises an organic solvent. In a preferred
embodiment, the organic solvent comprises tert-butyl alcohol,
iso-propyl alcohol, iso-butyl alcohol, or a mixture thereof.
[0170] In one embodiment of the aqueous process, the arabinoxylan
is separated according to the methods herein and then dissolved in
water. More preferably, the arabinoxylan is utilized directly from
the concentrated liquid or the liquid comprising arabinoxylan
without prior separation. In this case, the reaction medium should
be sufficiently alkaline to allow O-alkylation. If necessary,
additional alkaline material may be added to provide a pH of
greater than about 10. If the arabinoxylan has been separated and
washed to remove alkaline extractant, in one embodiment, it is
necessary to add an alkali metal hydroxide to the O-alkylation
reaction mixture in order to render the reaction medium alkaline.
NaOH and KOH, or a mixture thereof are the particularly preferred
alkali metal hydroxides.
[0171] It is preferred that the O-alkylation reaction is conducted
at from about 50.degree. C. to about 180.degree. C., preferably at
atmospheric pressure. In a further preferred embodiment, elevated
pressure may be used. Such elevated pressures may be from about 5
psi to about 100 psi, more preferably from about 5 psi to about 40
psi. In an additional embodiment, the temperature of the
O-alkylating agent contacting step is from about 50.degree. C. to
about 180.degree. C. and the O-alkylating agent contacting step is
conducted for from about 0.5 h to about 24 h.
[0172] In another, still preferred embodiment, the reaction medium
is non-aqueous. Further preferably, the reaction medium is aqueous.
Still further preferably, the non-aqueous or aqueous reaction media
further comprise an organic solvent. It is preferred that the
organic solvent comprises tert-butyl alcohol, iso-propyl alcohol,
iso-butyl alcohol, or a mixture thereof. In one embodiment, an
alkaline material is added to non-aqueous or aqueous reaction media
comprising an organic solvent. The O-alkylating agent contacting
step is conducted at from about 50.degree. C. to about 180.degree.
C. at a pressure of from about atmospheric to about 100 psi. More
preferably, the O-alkylating agent contacting step is conducted at
from about 80.degree. C. to about 120.degree. C. at a pressure of
about 5 to about 40 psi.
[0173] A further preferred embodiment relates to the type of
O-alkylating agent utilized to prepare the arabinoxylan ethers
herein. In one embodiment, the O-alkylating agent comprises
alkylene oxide, aryl substituted alkylene oxide, halogen
substituted alkylene oxide, alkyl halide, hydroxyalkyl halide, aryl
alkyl halide, carboxyalkyl halide, (alkyloxycarbonyl)alkyl halide,
allyl halide, vinyl halide, alkyl sulfonate, hydroxyalkyl
sulfonate, aryl alkyl sulfonate, carboxyalkyl sulfonate,
(alkyloxycarbonyl)alkyl sulfonate, allyl sulfonate, or a mixture
thereof. In a further preferred embodiment, the O-alkylating agent
comprises ethylene oxide, propylene oxide, butylene oxide,
epoxybutene, amylene oxide, glycidol, styrene oxide,
epichlorohydrin, methyl chloride, methyl iodide, methyl bromide,
ethyl bromide, propyl bromide, butyl bromide, propyl methyl
sulfonate, methyl chloroacetic acid, ethyl chloroacetic acid,
sodium chloroacetate, chloroacetic acid, benzyl bromide,
1-N,N-dialkylamino-2-chloroethane, or a mixture thereof. In a
further, particularly preferred embodiment, the O-alkylating agent
comprises ethylene oxide, propylene oxide, epoxybutene, methyl
chloride, ethyl bromide, sodium chloroacetate, or a mixture
thereof. In a most preferred embodiment, the O-alkylating agent
comprises epoxybutene.
[0174] In yet a further preferred embodiment, the arabinoxylan
ether consists essentially of an etherfied hemicellulose B. Yet
further, the arabinoxylan ether has a DS/CU of from about 0.01 to
about 2.5. Still further, the arabinoxylan ether has a DS/CU of
from about 0.3 to about 2.2. In an additional preferred embodiment,
the arabinoxylan ether has a MS/CU of from about 0.01 to about 100.
In yet a further preferred embodiment, the arabinoxylan ether has a
MS/CU of from about 0.1 to about 5.
[0175] Novel arabinoxylan ethers are also prepared according to the
methods herein. Such arabinoxylan ethers are prepared as the
product of the reaction of the separated arabinoxylan obtained
according to the methods herein with a C1 to C10 O-alkylating
agent. In one embodiment, the novel arabinoxylan ether is prepared
from an essentially branched and water soluble arabinoxylan
consisting essentially of an arabinoxylan of a weight average
molecular weight of greater than about 300,000. Still further, the
novel arabinoxylan ethers of this invention are prepared from an
arabinoxylan comprised of a xylan main polymer chain with at least
two branches, wherein the branches comprise groups of xylose,
arabinose, galactose, glucuoronic acid, 4-O-methyl glucouronic
acid, or a mixture thereof. Yet still further, the novel
arabinoxylan ethers are prepared from an arabinoxylan which is
soluble in water at a pH of from about 1 to about 14.
[0176] Still further the invention provides arabinoxylan ethers
prepared by the methods herein.
[0177] In a next major embodiment, the invention provides a method
of obtaining at least one monosaccharide from corn fiber comprising
the steps of: (a) heating an aqueous mixture of corn fiber and a
liquid; (b) contacting the mixture of step (a) with a protease
enzyme, thereby providing a proteolyzed corn fiber and a liquid;
(c) separating the liquid from the proteolyzed corn fiber; (d)
contacting the proteolyzed corn fiber at least once with an alkali
extractant, thereby providing an insoluble cellulose material and a
liquid comprising arabinoxylan; (e) separating the insoluble
cellulose material from the liquid comprising arabinoxylan; (f)
reducing the volume of the liquid comprising arabinoxylan by
removing excess alkaline extractant, thereby providing a
concentrated liquid comprising from about 10 to about 50% solids
wherein the solids comprise an arabinoxylan; and (g) hydrolyzing
the arabinoxylan of step (f) in the presence of a catalyst and a
solvent, thereby providing a mixture comprising at least one
monosaccharide.
[0178] The proteolyzed corn fiber utilized in this aspect of the
invention may be destarched and/or solvent extracted as disclosed
above. In one embodiment, the alkaline extractant comprises the
alkaline extractant utilized in the extraction methods set out
above. In a further embodiment, the arabinoxylan may be separated
from the concentrated liquid or the liquid comprising arabinoxylan
according to the methods set out above. However, in order to
increase the industrial utility of the process, it is particularly
preferred that the hydrolysis be conducted while the arabinoxylan
is still in the concentrated liquid or the liquid comprising
arabinoxylan.
[0179] In a preferred embodiment, the volume reducing step is
conducted via ultrafiltration as described above prior to
contacting the arabinoxylan with a catalyst. This process allows
the separate recovery of alkaline extractant so that less acid is
needed to neutralize the excess alkaline extractant. Further, since
the arabinoxylan is preferably not isolated as a precipitate prior
to hydrolysis, the arabinoxylan will be already dissolved in water,
which is a preferred solvent for converting the arabinoxylan to at
least one monosaccharide.
[0180] Moreover, in the prior treatments set out above, many of the
undesirable components that would otherwise be extracted from corn
fiber, eg. glucose, have been removed. Hence, hydrolysis of a
concentrated form of the arabinoxylan allows more effective
separation of desirable monosaccharides than could otherwise be
obtained from corn fiber not treated in accordance with the methods
herein. Those skilled in the art will recognize that these factors
combine to provide a more economical route to obtain these valuable
monosaccharides than could otherwise be achieved.
[0181] Further, in a surprising finding in the invention herein,
corn fiber arabinoxylan isolated by precipitation in acetic acid
can be carried directly to the hydrolysis step without needing to
remove the acetic acid from the arabinoxylan. The acetic acid can
have a beneficial effect on the hydrolysis and, as such, the
hydrolysis mixture can be taken on to the subsequent monosaccharide
separation steps without the necessity of removing the acetic acid.
Even more surprisingly, as is set out below, the arabinoxylan
precipitate containing acetic acid can be carried forward to the
epimerization of L-arabinose to L-ribose. As those of ordinary
skill in the art will recognize, these findings provide highly
beneficial results in an industrial process. However, as noted, it
is not necessary to first precipitate the arabinoxylan prior to
conducting the hydrolysis steps.
[0182] In a preferred embodiment, the catalyst of step (g) is an
inorganic acid and the solvent is water. In a further preferred
embodiment, the acid is added in an amount suitable to result in a
pH of less than about 2.0. Still further, in a preferred
embodiment, the acid may comprise H.sub.2SO.sub.4, HCl, or a
mixture thereof. Yet further preferably, the amount of inorganic
acid is from about 1.0 wt. % to about 30 wt. % in excess of that
required to neutralize any residual alkaline extractant in the
concentrated liquid, as measured by dry weight of arabinoxylan. In
a further embodiment, step (g) further comprises acetic acid.
[0183] In a preferred embodiment, the arabinoxylan obtained from
step (g), whether or not first separated from the concentrated
liquid, is heated to from about 70.degree. C. to about 120.degree.
C. for from about 0.1 h to about 24 h, thereby providing a mixture
of monosaccharides, wherein the mixture of monosaccharides
comprises at least about 70 wt. % of L-arabinose and D-xylose.
[0184] As used here, and with regard to other separate embodiments
of the monosaccharide separation methods, the 70 wt. % figure
denotes the total amount of the specified monosaccharides in the
total mixture of monosaccharides. The balance of the wt. % totaling
100% comprises other materials that relate to the remainder of the
components present in the arabinoxylan. As an example, in one
embodiment, the arabinoxylan extracted from corn fiber is comprised
of a xylan main polymer chain with at least two branches, wherein
the branches comprise groups of xylose, arabinose, galactose,
glucouronic acid, 4-O-methyl-glucouronic acid, or a mixture
thereof. Thus, the 30 wt. % not constituting L-arabinose and
D-xylose, in one embodiment, is comprised of the other materials
within the arabinoxylan in corn fiber such as galactose,
glucouronic acid, etc.
[0185] In a further embodiment, the L-arabinose and D-xylose are
separately isolated from the mixture of monosaccharides as
individual fractions, thereby separately providing a L-arabinose
fraction and a D-xylose fraction. As used herein, "separately
providing" means that at least two separate fractions are
individually provided, wherein subsequent processing steps may be
conducted with a single fraction, but not the other, because the
fractions are not combined.
[0186] Although a number of methods can be used for separation of
the monosaccharide materials, a particularly preferred method of
separation herein involves a technique known as "simulated moving
bed ("SMB") chromatography." SMB chromatography is a binary
separation technique which can be used to separate a feed mixture
into two fractions. For example, a feed mixture comprised of a
xylose and arabinose mixture will be split into two fractions, one
enriched in xylose and the other enriched in arabinose. Any
impurity will end up with either the xylose fraction or the
arabinose fraction. The two fractions can be further purified by
crystallization, ultrafiltration, further SMB chromatography or
other methods known to one of ordinary skill in the art.
[0187] If the feed contains three components which are all
valuable, a two-stage SMB chromatographic process is generally
utilized. The first stage splits the ternary feed into a fraction
enriched in one component and a fraction enriched in the other two
components. The second fraction can then be split into two
fractions, each enriched in one of the two components. However, it
is possible to separate a ternary mixture into three fractions
using a single SMB chromatographic technique. (See Navarro et al.;
Journal of Chromatography A, 770 (1997) 39-50, the disclosure of
which is incorporated herein in its entirety.)
[0188] The SMB chromatographic unit preferably utilized herein
preferably consists of either a multi-sectioned column or multiple
columns. The feed mixture introduced to the SMB chromatographic
unit is split into two fractions, referred to as "raffinate" and
"extract" streams. A liquid desorbant is also fed to the unit; this
material acts as a regenerant. The two inlet ports and the two
outlet ports are moved intermittently to a next section in a
multi-section column or to a next section column in a multi-column
unit in the direction of liquid flow. The movement of the ports
simulates the countercurrent movement of the packing. An effective
movement of the packing in the direction opposite to the liquid
flow results in the separation of the feed components into the
separate fractions.
[0189] Because of the expense and difficulty of exploring SMB
chromatographic separations on large pilot plant equipment,
laboratory experiments have been developed which predict the
separation of two components using SMB chromatography. One such
experiment is a pulse test which, when performed on larger columns,
directly provides preliminary design data for the SMB
chromatographic unit. In this experiment, a column is packed with
the required resin and the sample is introduced as a pulse which is
immediately followed by eluent while maintaining a constant flow
rate. Samples of the eluents from the column are collected and
analyzed. (Balannec, B. and Hotier, G., "From Batch Elution to
Simulated Countercurrent Chromatography," in "Preparative and
Production Scale Chromatography," Genetsos, G. and Baker, P. E.,
editors. Chromatographic Science Series Volume 61, Marcel Dekker,
Inc. 1992, the disclosure of which is included herein in its
entirety.)
[0190] In a preferred embodiment of the invention herein, a
hydrolysis mixture which contains at least two components wherein
at least one of the components comprises a monosaccharide is
treated to remove salts prior to commencing the SMB chromatography.
It is further preferred that the salts comprise sulfate salts which
are removed from the mixture of step (g) comprising at least two
components via precipitation with CaCO.sub.3 or Ca(OH).sub.2. Still
further preferred, the mixture containing at least one
monosaccharide is passed through an ion exchange column capable of
removing salts arising from an inorganic acid catalyst. Yet further
preferred is the use of ion exclusion-SMB chromatography to remove
the salts from the mixture comprising at least two components to
recover sulfuric acid from the neutral materials. Yet even further
preferred, the salts are removed from the mixture comprising at
least two components by electrodialysis.
[0191] In yet another preferred embodiment, SMB is used to separate
at least two components from the hydrolysis mixture of step (g)
into two fractions. Water is preferably used at the eluent. It is
preferred that the concentration of the feed be between from about
5 wt. % to about 85 wt. % and more preferably, from about 35 wt. %
to about 65 wt. %. It is further preferred that the temperature of
the eluent and feed be from about 25.degree. C. to about 80.degree.
C. and, more preferably, from about 40.degree. C. to about
65.degree. C. Those skilled in the art will recognize that the
upper limit of monosaccharide concentration in the feed mixtures
will be monosaccharide solubility at the maximum operating
temperature.
[0192] In a preferred embodiment, the mixture fed onto the SMB
comprises from about 5 wt. % to about 85 wt. % of a mixture of
D-xylose and L-arabinose. Still further, the at least two
components fed onto the SMB comprise D-xylose and L-arabinose.
Still further, after the separation step L-arabinose is present in
one eluent fraction at from about 85 wt. % to about 100 wt. %.
Still further preferably, minor components, eg., galactose, are
allowed to go into the D-xylose fraction, thereby providing an
L-arabinose fraction comprised from about 85 wt % to about 100 wt %
L-arabinose. In a separate embodiment, minor components, eg.,
galactose, are allowed to go with the L-arabinose fraction, thereby
providing a D-xylose fraction comprised from about 85 wt. % to
about 100 wt. % D-xylose. Still further, it is preferred that the
D-xylose fraction is comprised of from about 85 wt. % to about 100
wt. % D-xylose. Those skilled in the art will recognize that these
fractions can be additionally purified by crystallization.
[0193] In yet another preferred embodiment, a resin is utilized in
the SMB separation and the resin is a cation resin exchanged with a
salt selected from the group consisting of calcium or magnesium,
with calcium being particularly preferred. Examples of preferred
resins include, but or not limited to, Dowex Monosphere 99 (Dow
Chemical, Midland, Mich.), Rohm and Haas CR1320 (Philadelphia,
Pa.), Purolite PCR-642 (Purolite Company, Philadelphia, Pa.), and
Mitsubishi (Mitsubishi Chemical, White Plains, N.Y.).
[0194] In yet another preferred embodiment, the resin utilized for
SMB chromatographic separation is a strongly basic resin in a
phosphate form. An example, but not limited to, of a preferred
resin is Dowex-1 (Dow Chemical, Midland, Mich.) strongly basic
resin in the phosphate form.
[0195] In a separate, still preferred embodiment, the materials of
hydrolysis step (g) are heated to from about 40.degree. C. to about
70.degree. C. for from about 0.1 h to about 24 h in the presence of
an inorganic acid catalyst, thereby providing a mixture of
monosaccharides and xylan, wherein the mixture of monosaccharides
comprises at least about 50 wt. % L-arabinose. The xylan and the
mixture of monosaccharides comprising L-arabinose may be separately
isolated to provide a xylan precipitate and a mixture of
monosaccharides in a preferred embodiment by adding a solvent
comprising methanol, ethanol, n-propyl alcohol, isopropyl alcohol,
or a mixture thereof. In yet a further embodiment, the xylan
precipitate may be separated from the mixture of monosaccharides
comprising at least about 50 wt. % L-arabinose via ultrafiltration.
In a further preferred embodiment, the L-arabinose may be separated
from the mixture of monosaccharides to separately provide a
L-arabinose fraction and a mixture of monosaccharides according to
the SMB method discussed above.
[0196] In a further preferred method, the invention comprises the
additional steps of: dispersing the xylan precipitate in a suitable
solvent, such as water, thereby providing a xylan solution; adding
from about 1 wt. % to about 30 wt. % of a catalyst to the solution;
and adjusting the temperature of the solution to from about
70.degree. C. to about 120.degree. C. for from about 0.1 h to about
24 h, thereby providing a mixture of monosaccharides, wherein the
mixture of monosaccharides comprises at least 50% D-xylose. In this
embodiment, the catalyst may comprise H.sub.2SO.sub.4, HCl, acetic
acid, or a mixture thereof. Still further, the D-xylose may be
isolated from the mixture of monosaccharides to separately provide
a D-xylose fraction and a mixture of monosaccharides. One method of
separation is preferably SMB chromatography as set out above.
[0197] In a separate preferred embodiment, xylan is separated from
the mixture of monosaccharides via ultrafiltration utilizing a
xylan-rejecting membrane, thereby providing a xylan solution and a
mixture of monosaccharides wherein the mixture of monosaccharides
comprises at least about 50 wt. % L-arabinose. When ultrafiltration
is utilized, the xylan obtained is preferably a precipitate. In a
further preferred embodiment, the L-arabinose may be separated from
the mixture of monosaccharides according to the SMB methods
discussed above, thereby separately providing an L-arabinose
fraction and a mixture of monosaccharides.
[0198] One of ordinary skill in the art will recognize the
techniques that may be utilized in the ultrafiltration methods
herein. In particular, one of ordinary skill in the art would
realize that the particle size/molecular weight of the
monosaccharides in the mixture of monosaccharides may be separately
determined and the results utilized to select the appropriate
membrane to allow monosaccharides to pass through the membrane,
while leaving xylan or other specified monosaccharides on a front
side of the membrane and vice versa.
[0199] In a further preferred embodiment, the invention comprises
the additional steps of: adding from about 1 wt. % to about 30 wt.
% of a catalyst to the xylan solution; and adjusting the
temperature of the xylan solution to from about 70.degree. C. to
about 120.degree. C. for from about 0.1 h to about 24 h, thereby
providing a mixture of monosaccharides, wherein the mixture of
monosaccharides comprises at least about 50 wt. % D-xylose. In this
embodiment, the catalyst may comprise H.sub.2SO.sub.4, HCL acetic
acid, or a mixture thereof. Still further, the D-xylose is
separated from the mixture of monosaccharides, thereby separately
providing a D-xylose fraction and a mixture of monosaccharides. A
preferred method of separation of the D-xylose is via SMB
chromatography as set out above.
[0200] In yet a further preferred embodiment, the catalyst of step
(g) of the method of obtaining at least one monosaccharide from
corn fiber comprises at least one enzyme and the solvent comprises
water. Still further preferably, the pH of step (g) is adjusted to
from about 4 to about 9 prior to the addition of the enzyme. The
enzyme may preferably comprise arabinofuranosidase. An example of
such an enzyme can be found in U.S. Pat. No. 5,882,905, the
disclosure of which is incorporated herein by this reference in its
entirety. In a further, still preferred embodiment, the solvent may
comprise water and the catalyst may comprise both an
arabinofuranosidase enzyme and a xylanase enzyme. In a particularly
preferred embodiment, the arabinofuranosidase enzyme is added to
the xylan solution prior to the addition of the xylanase
enzyme.
[0201] In the enzyme catalyst method, the materials of step (g) are
heated to from about 25.degree. C. to about 90.degree. C. for from
about 0.1 h to about 24 h, thereby providing a mixture of
monosaccharides comprising at least about 70 wt. % of a mixture
L-arabinose and D-xylose. Still further, in a preferred embodiment,
the method comprises the step of isolating as separate fractions
the mixture of L-arabinose and the D-xylose from the mixture of
monosaccharides. Still further, the method preferably comprises
separately isolating the L-arabinose and D-xylose from each other,
thereby providing an individual L-arabinose fraction and an
individual D-xylose fraction. The method of separation may be
according to the SMB method as discussed herein.
[0202] In yet further preferred embodiments of the invention
herein, the materials in step (g) are heated to from about
25.degree. C. to about 90.degree. C. for from about 0.1 h to about
24 h, thereby providing a mixture of monosaccharides and xylan,
wherein the mixture of monosaccharides comprises at least 50 wt. %
L-arabinose. Still further, a solvent comprising methanol, ethanol,
n-propyl alcohol, isopropyl alcohol, or a mixture thereof may
preferably be added to the mixture of monosaccharides and xylan,
thereby providing a xylan precipitate and a mixture of
monosaccharides, wherein the mixture of monosaccharides comprises
at least 50 wt. % L-arabinose. In yet a further embodiment, the
xylan precipitate is separately isolated from the mixture of
monosaccharides comprising at least about 50 wt. % L-arabinose. In
a further preferred embodiment, the L-arabinose may be separated
from the mixture of monosaccharides according to the SMB method
discussed above, thereby providing a L-arabinose fraction and a
mixture of monosaccharides.
[0203] In yet a further preferred embodiment, the method comprises
the additional steps of: dispersing the xylan precipitate in a
suitable solvent to provide a xylan solution; adding from about 1
wt. % to about 30 wt. % of a catalyst to the xylan solution; and
adjusting the temperature to from about 70.degree. C. to about
120.degree. C. for from about 0.1 h to about 24 h, thereby
providing a mixture of monosaccharides wherein the mixture
comprises at least 50% D-xylose. In this preferred embodiment, the
catalyst comprises H.sub.2SO.sub.4, HCl, acetic acid or a mixture
thereof. In a still further preferred embodiment, the method
comprises separating the D-xylose from the mixture of
monosaccharides to provide an individual D-xylose fraction and a
mixture of monosaccharides. The separation may be utilizing the SMB
method discussed above.
[0204] In a separate preferred embodiment, xylan is separated from
the mixture of monosaccharides via ultrafiltration, according to
the methods set out above, utilizing a xylan-rejecting membrane,
thereby providing a xylan solution and a mixture of
monosaccharides, wherein the mixture of monosaccharides comprises
at least about 50 wt. % L-arabinose. In a further preferred
embodiment, the L-arabinose may be separated from the mixture of
monosaccharides according to the SMB methods discussed above,
thereby separately providing a L-arabinose fraction and a mixture
of monosaccharides.
[0205] In a further preferred embodiment, the method comprises the
additional steps of: adding from about 1 wt. % to about 30 wt. % of
a catalyst to the xylan solution prepared above; and adjusting the
temperature of the xylan solution to from about 70.degree. C. to
about 120.degree. C. for from about 0.1 h to about 24 h, thereby
providing a mixture of monosaccharides, wherein the mixture
comprises at least about 50 wt. % D-xylose. In this embodiment, the
catalyst comprises H.sub.2SO.sub.4, HCl, acetic acid or a mixture
thereof. Still further, the D-xylose is separated as an individual
fraction from the mixture of monosaccharides. The preferred method
of separation of the D-xylose is via SMB chromatography as set out
above.
[0206] In a preferred embodiment, the catalyst of hydrolysis step
(g) is an inorganic acid and the solvent is acetic anhydride. In a
further preferred embodiment, the acid is added to result in a pH4
of less than about 2.0. Still further, in a preferred embodiment,
the acid may comprise H.sub.2SO.sub.4, HCl, or a mixture thereof.
Yet further preferably, the amount of inorganic acid is from about
1.0 wt. % to about 30 wt. % in excess of that required to
neutralize residual alkaline extractant, as measured by dry weight
of arabinoxylan.
[0207] In a preferred embodiment, the mixture of step (g) may be
heated to a temperature of from about 70.degree. C. to about
120.degree. C. for from about 0.1 h to about 24 h in the presence
of the inorganic acid and acetic anhydride, thereby providing a
mixture of monosaccharides acetates wherein the mixture of
monosaccharide acetates comprises at least 70% of a mixture of
L-arabinose acetate and D-xylose acetate. In this embodiment,
xylose or arabinose acetate means any acetyl ester of xylose or
arabinose which may be in either the pyranose or furanose form. The
mixture of D-xylose acetate and L-arabinose acetate may preferably
be isolated from the mixture of monosaccharides as individual
fractions. Still further, the D-xylose acetate and L-arabinose
acetate may be separately isolated from each other. The preferred
means of separation is SMB chromatography.
[0208] Yet still further preferably, the mixture of L-arabinose
acetate and D-xylose acetate may be contacted with a hydrolyzing
agent for a time and at a temperature sufficient to provide a
mixture of L-arabinose and D-xylose. The L-arabinose and D-xylose
may be preferably isolated as individual fractions. In a preferred
embodiment, the hydrolyzing agent is NaOMe in MeOH or aqueous
NH.sub.4. The contacting may preferably be conducted at a
temperature of from about 25.degree. C. to about 65.degree. C. for
from about 0.1 h to about 24 h. Still further preferably, the
contacting temperature is from about 25.degree. C. to about
65.degree. C. for from about 0.1 h to about 24 h.
[0209] In a further preferred embodiment, the L-arabinose acetate
may separately be hydrolyzed with NaOMe in MeOH or aqueous NH.sub.4
to provide L-arabinose. Still further, the isolated D-xylose
acetate may preferably be separately hydrolyzed with NaOMe in MeOH
or aqueous NH.sub.4 to provide D-xylose.
[0210] In yet a further embodiment, the mixture of step (g) is
preferably heated to a temperature of from about 40.degree. C. to
about 65.degree. C. for from about 0.1 h to about 24 h, thereby
providing a mixture of monosaccharide acetates and xylan acetate
wherein the mixture of monosaccharide acetates comprises at least
about 20 wt. % L-arabinose acetate. Still further, methanol,
ethanol, n-propyl alcohol, isopropyl alcohol or aqueous acetic acid
may be added, thereby providing a precipitate comprising xylan
acetate and a mixture of monosaccharide acetates wherein the
monosaccharide acetates comprises at least about 20 wt. %
L-arabinose acetate. In yet a further embodiment, the xylan acetate
precipitate is separated from the mixture of monosaccharide
acetates comprising at least about 20 wt. % L-arabinose acetate via
an ultrafiltration method. In a further preferred embodiment, the
L-arabinose acetate may be separated from the mixture of
monosaccharide acetates according to the SMB methods discussed
above, thereby providing a L-arabinose acetate fraction and a
mixture of monosaccharides.
[0211] In yet a further preferred embodiment, the method comprises
the additional steps of: dispersing the xylan acetate precipitate
in a suitable solvent, such as water, to provide a xylan acetate
solution; adding from about 1 wt. % to about 30 wt. % of a catalyst
to the xylan acetate solution; and adjusting the temperature to
from about 70.degree. C. to about 120.degree. C. for from about 0.1
h to about 24 h thereby providing a mixture of monosaccharides,
wherein the mixture of monosaccharides comprises at least 50%
D-xylose. In this preferred embodiment, the catalyst comprises
H.sub.2SO.sub.4, HCl, acetic acid or a mixture thereof. In a still
further preferred embodiment, the method comprises separating the
D-xylose from the mixture of monosaccharides, thereby providing a
D-xylose fraction and a mixture of monosaccharides. The separation
may be utilizing the SMB method discussed above.
[0212] In yet a further preferred embodiment, the method of
obtaining at least one monosaccharide from corn fiber comprises
isolating xylan acetate via ultrafiltration through a xylan
acetate-rejecting membrane, thereby providing a xylan acetate
solution and a second solution of monosaccharide acetates
comprising at least 20 wt. % L-arabinose acetate. In a further
preferred embodiment, the L-arabinose acetate may be isolated from
the mixture of monosaccharide acetates according to the SMB methods
discussed above, thereby providing a L-arabinose fraction.
[0213] Yet further preferably, the isolated L-arabinose acetate is
hydrolyzed with a NaOMe/MeOH solution or an aqueous NH.sub.4
solution to provide a L-arabinose fraction. The hydrolysis is
preferably conducted at a temperature of from about 25.degree. C.
to about 65.degree. C. for from about 0.1 h to about 24 h. Still
further, the isolated xylan acetate is hydrolyzed with a NaOMe/MeOH
solution or an aqueous NH.sub.4 solution to provide a D-xylose
fraction. The hydrolysis is preferably conducted at a temperature
of from about 25.degree. C. to about 65.degree. C. for from about
0.1 h to about 24 h.
[0214] In yet a further preferred embodiment of the invention
herein, the xylan acetate is contacted with water and an acid at a
temperature of from about 70.degree. C. to about 120.degree. C. for
from about 0.1 h to about 24 h, thereby providing a mixture of
monosaccharides comprising at least 50% D-xylose. The acid may
preferably be H.sub.2SO.sub.4, HCl, or a mixture thereof. Yet still
further preferably, the D-xylose maybe isolated from the mixture of
monosaccharides as a individual fraction.
[0215] In a next embodiment, the invention provides a method of
obtaining at least one monosaccharide from corn fiber comprising
the steps of: (a) heating an aqueous mixture of corn fiber and a
liquid; (b) contacting the mixture of step (a) with a protease
enzyme, thereby providing a proteolyzed corn fiber and a liquid;
(c) separating the liquid from the proteolyzed corn fiber; (d)
contacting the proteolyzed corn fiber at least once with an alkali
extractant, thereby providing an insoluble cellulose material and a
liquid comprising arabinoxylan; (e) separating the insoluble
cellulose material from the liquid comprising arabinoxylan; (f)
reducing the volume of the liquid comprising arabinoxylan by
removing excess alkaline extractant, thereby providing a
concentrated liquid comprising from about 10 to about 50% solids,
wherein the solids comprise an arabinoxylan; (g) hydrolyzing the
arabinoxylan in the presence of a catalyst and a solvent, thereby
providing a mixture comprising at least one monosaccharide; and (h)
separating at least one monosaccharide from the mixture of
monosaccharides.
[0216] In a preferred embodiment of the method of obtaining at
least one monosaccharide from corn fiber, the at least one
monosaccharide comprises L-arabinose and the method further
comprises contacting the L-arabinose with a solvent and a Mo(VI)
catalyst at a temperature and a pH sufficient to provide a mixture
of L-arabinose and L-ribose. In this preferred method, at least
some of the L-arabinose is epimerized to L-ribose. In a further
embodiment, the solvent comprises water. In yet another embodiment,
the solvent comprises a mixture of water and acetic acid. In a
further embodiment, the concentration of acetic acid is from about
5 wt. % to about 90 wt. % based upon a total volume of acetic acid
and water. Still further, the concentration of acetic acid is from
about 20 wt. % to about 80 wt. % based upon a total volume of
acetic acid and water. The preferred concentration of catalyst is
from about 0.01 wt. % to about 10 wt. %, as measured by dry weight
of L-arabinose. Yet further preferably, the concentration of
catalyst is from about 0.5 to about 2 wt. %, as measured by dry
weight of L-arabinose.
[0217] In this epimerization of arabinose and xylose, it has been
surprisingly found that utilizing acetic acid as a solvent with
Mo(VI) as the catalyst effectively promotes epimerization of
arabinose to ribose and xylose to ribose. This is an unexpected
result because one would anticipate that the acetic acid would, in
fact, promote degradation of the arabinose and/or xylose. Further,
it would be expected that acetic acid would deactivate the Mo(VI)
catalyst. However, one of ordinary skill in the art will recognize
that it is not necessary to first separate the arabinoxylan from
the concentrated liquid in order to conduct this epimerization. In
fact, it will be apparent that in some circumstances it is not
desirable to perform such separations.
[0218] In a further embodiment, the concentration of L-arabinose in
the mixture is from about 10 wt. % to about 70 wt. %. Still
further, the concentration of L-arabinose in the mixture is from
about 40 wt. % to about 60 wt. %. A preferred pH for the hydrolysis
step is from about 1 to about 4.5, more preferably from about 2.5
to about 3.0. In a particularly preferred embodiment, the
hydrolysis step is conducted at a temperature of from about
80.degree. C. to about 100.degree. C., or, at a still preferred
temperature of from about 20.degree. C. to about 100.degree. C.
Still further preferred is when the hydrolysis temperature is from
about 60.degree. C. to about 100.degree. C. A preferred contact
temperature is from about 20.degree. C. to about 100.degree. C.
Still further preferred is when the hydrolysis temperature is from
about 60.degree. C. to about 100.degree. C. The preferred
hydrolysis time is from about 0.1 to about 20 h and, even more
preferred from about 1 to about 5 h and, still more preferred, from
about 2 to about 4 h.
[0219] Still further, it is preferred that the Mo(VI) catalyst is
ammonium dimolybdate, sodium molybdate, MoO.sub.3, or a mixture
thereof. In a further embodiment, the catalyst is a solid catalyst
obtained by heating ammonium dimolybdate in water at a sufficient
pH, temperature and time to cause the precipitation of a
polymolybdate from the aqueous solution. One of ordinary skill in
the art will recognize that such conditions may be determined
without undue experimentation. In a preferred embodiment, the solid
catalyst is isolated and utilized in the hydrate form. Still
further preferably, the catalyst is a solid catalyst obtained by
heating ammonium dimolybdate in water at a pH of from about 1 to
about 3 at from about 80.degree. C. to about 100.degree. C. for
from about 2 to about 5 h.
[0220] In yet a further embodiment, the concentration of the
catalyst is from about 0.01 wt. % to about 10 wt. %. It is further
preferred that the concentration of the catalyst is from about 0.5
wt. % to about 2 wt. %. Still further, the catalyst is preferably
deposited on an immobile solid support.
[0221] It is preferred that the L-ribose obtained in this aspect of
the invention be isolated from the L-arabinose as an individual
fraction, thereby separately providing a L-arabinose fraction and a
L-ribose fraction. The L-ribose may be isolated vial SMB
chromatography, wherein a L-ribose eluent fraction and a
L-arabinose eluent fraction are separately provided. In this
method, it is preferred that the L-arabinose eluent fraction is
contacted one or more times with a Mo(VI) catalyst at a time,
temperature and pH suitable to provide further L-ribose.
[0222] One of ordinary skill in the art will recognize that the
particular combination of pH, temperature and concentration of
catalyst selected can impact the time required for converting
L-arabinose to a mixture of L-arabinose and L-ribose. It should
also be recognized that ultimately an equilibrium mixture of
L-arabinose and L-ribose is obtained so that prolonged contact
times are not believed to provide additional benefit and may, in
fact, result in decomposition of the monosaccharides.
[0223] In a most preferred embodiment, the solution of L-arabinose
utilized for the epimerization reaction is that obtained by SMB
chromatographic separation of a D-xylose fraction and a L-arabinose
fraction from the hydrolysis of arabinoxylan according to the
methods herein.
[0224] The preferred amount of L-ribose fraction in the mixture of
the L-arabinose fraction and L-ribose fraction after contact with
the catalyst is from about 1 wt. % to about 30 wt. % based on the
combined dry weight of L-arabinose and L-ribose. Still further
preferably, the amount of L-ribose in the mixture of L-arabinose
and L-ribose after contact with the catalyst is from about 20 wt. %
to about 25 wt. % based on the combined dry weight of L-arabinose
and L-ribose.
[0225] In yet a further embodiment, the L-arabinose and L-ribose
are separated from each other by SMB chromatography as set out
above to provide individual solutions. In yet a further preferred
embodiment, the solution of separated L-arabinose is returned to
the reaction vessel one or more times for further contact with the
Mo(VI) catalyst under conditions sufficient to cause conversion of
the L-arabinose to a mixture of L-arabinose and L-ribose, whereby a
further separation of L-ribose is conducted. One of ordinary skill
in the art will recognize that such a method maximizes the yield of
L-ribose. In a particularly preferred process, the epimerization
reaction is conducted in a continuous process. Still further
preferably, the reaction vessel contains the polymolybdate catalyst
described above deposited on an immobile solid support.
[0226] In yet a further embodiment, the method of obtaining
monosaccharides from corn fiber further comprises the additional
steps of: contacting the mixture of monosaccharides with a xylose
reductase, thereby providing a mixture of xylitol and L-arabinose;
and dispersing the xylitol in a solvent in which L-arabinose does
not dissolve, thereby providing a xylitol/solvent solution and an
L-arabinose precipitate. In a particularly preferred embodiment,
the xylose reductase enzymes utilized herein are substantially free
of arabinose reductase activity. The preferred xylose reductases
exhibit a wide range of activities and conditions under which they
exhibit maximum activity. In one embodiment, the xylose reductase
treatment is conducted at a temperature of from about 20.degree. C.
to about 100.degree. C., more preferably between about 40.degree.
C. to about 70.degree. C. at a pH of from about 5 to about 8.
Reaction times can range from between 0.1 to about 20 hours,
preferably between about 1 to about 3 hours. Those of skill in the
art will recognize that the exact conditions utilized for this, as
well as the other enzyme treatments herein, will vary due to the
source and, thus, the specific characteristics of the enzyme
illustrated.
[0227] In a next major aspect, the invention comprises a method of
obtaining soluble proteins and carbohydrates from corn fiber
comprising the steps of: heating an aqueous suspension of corn
fiber; contacting the fiber sequentially or concurrently with an
amylase enzyme and protease enzyme for a time and at a temperature
sufficient to provide an essentially destarched, proteolyzed corn
fiber and a liquid(s) comprising soluble proteins, carbohydrates,
or a mixture thereof; and separating the liquid from the destarched
corn fiber, wherein the soluble proteins and carbohydrates are
suitable as feedstock for the production of animal feed, chemicals,
or polymers.
[0228] In yet a further major aspect of this invention, a method
for obtaining animal feed is provided, wherein the method comprises
the steps of: heating an aqueous suspension of corn fiber;
contacting the fiber sequentially or concurrently with an amylase
enzyme and protease enzyme for a time and at a temperature
sufficient to provide an essentially destarched, proteolyzed corn
fiber and a liquid(s) comprising soluble proteins, carbohydrates,
or a mixture thereof; separating the liquid from the destarched,
proteolyzed corn fiber; contacting the corn fiber at least once
with an alkali extractant to provide an insoluble cellulose
material and a liquid comprising arabinoxylan; separating the
insoluble cellulose material from the liquid comprising
arabinoxylan; adding a sufficient amount of a corn steep liquor to
the cellulose material; and removing water from the heterogeneous
mixture thereby providing an animal feed.
EXAMPLES
[0229] The following Examples are set forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how the compounds and methods claimed herein are
made, performed and evaluated, and are intended to be purely
exemplary of the invention and are not intended to limit the scope
of what the inventors regard as their invention. Efforts have been
made to ensure accuracy with respect to numbers (e.g., amounts,
temperature, etc.) but some errors and deviations should be taken
into account. Unless indicated otherwise, parts are parts by
weight, temperature is in .degree. C. or is at room temperature,
and pressure is at or near atmospheric.
Example 1
Destarching of Corn Fiber
[0230] The following represents one method for destarching corn
fiber utilized according to the methods herein. The corn fiber
utilized either contained a low amount of moisture (about 10.4 wt.
% H.sub.2O) and no SO.sub.2 or the corn fiber was wet (about 65%
moisture) and contained SO.sub.2 (about 852 ppm sulfur). Unless
otherwise noted, the same corn fiber was utilized in the subsequent
Examples.
[0231] A 2000 mL three-neck glass round bottom flask equipped with
a mechanical stirrer and a reflux condenser was charged with 300 g
of corn fiber, 1080 mL H.sub.2O and 120 mL of a 1 M phosphate
buffer solution. The mixture was heated to 100.degree. C. and held
at this temperature for 20 minutes. The mixture was cooled to the
appropriate temperature before addition of enzyme. When no starch
was detectable by iodine staining, the solids were isolated by
filtration, washed, and dried at 60.degree. C.
1TABLE 1 COMPARISON OF SAMPLE DESTARCHING TREATMENTS Reac-
units/100 tion g corn Time Temp % % % % % % Xyl/ Sample Enzyme
fiber h (.degree. C.) Yield.sup.1 Glu Xyl Gal Ara Man Ara 1 None
(CornFiber) 52.2 25.9 5.4 16.5 0.0 1.6 9 Type X-A 168000.sup.2 1.7
30 73.3 35.2 37.0 6.8 21.0 0.0 1.8 fungal from Aspergillus oryzae 3
Type II-A 724000.sup.2 3.5 30 68.9 35.9 36.5 6.9 20.6 0.0 1.8
Bacillus 4 Type II-A 1140.sup.2 72 30 75.1 39.4 33.8 5.9 20.7 0.1
1.6 Bacillus 5 Spezyme 297400.sup.3 0.25 60 69.1 36.5 35.6 6.12 1.7
0.1 1.6 AA-L 6 Spezyme 297400.sup.3 0.28 80 70.7 36.6 36.2 4.6 22.4
0.1 1.6 AA-L 7 Spezyme 50600.sup.3 0.75 80 72.7 35.9 35.7 6.2 22.1
0.1 1.6 AA-L 8 Spezyme 25300.sup.3 0.83 80 74.6 37.6 35.0 6.0 21.3
0.1 1.6 AA-L 9 Spezyme 25300.sup.3 0.88 80 79.6 36.8 34.9 6.3 21.9
0.1 1.6 AA-L Notes for Table 1: .sup.1Yield of destarched fiber
based upon dry weight of corn fiber. .sup.2One unit will liberate
1.0 mg of maltose from starch in 3 min., pH 6.9, 20.degree. C.
.sup.3The activity of thermostable amylase units (TAU) where 1 TAU
is defined as the quantity of enzyme that will dextrinize 1 mg of
starch per minute at pH 6.6 and 30.degree. C.
[0232] This Example illustrates that the thermophilic
.alpha.-amylase, Spezyme, exhibited a higher activity at a higher
temperature, relative to the other amylase varieties examined.
Thus, when Spezyme is utilized, less enzyme is required, reaction
times are shorter, and the reaction temperature is comparable to
the temperatures (about 75 to about 100.degree. C.) useful for
swelling the corn fiber prior to enzymatic detaching. As shown by
Samples 5 and 6, each of which utilized an excess of Spezyme in
order to ensure complete removal of starch, corn fiber contains
approximately 30% starch. Hence, under optimal conditions, yields
of from about 68% to about 73% of destarched corn fiber can be
expected. Under conditions in which all of the starch component has
been removed from the corn fiber, such as illustrated by Samples 3
and Samples 5-7, the glucose and xylose content of destarched corn
fiber is typically from about 35% to about 37% and the arabinose
content is from about 20% to about 23%, thereby providing a xyl/Arb
ratio of from about 1.8 to about 1.5.
Example 2
Corn Fiber Destarching Process
[0233] A 5000 mL three-neck glass round bottom flask equipped with
a mechanical stirrer and a reflux condenser was charged with 250 g
of corn fiber, 1800 mL H.sub.2O, and 200 mL of a 1 M phosphate
buffer solution. The mixture was heated to 80.degree. C., followed
by addition of 10 mL of a Spezyme suspension. Generally, using
iodine staining, no starch could be detected after 50 minutes.
[0234] In order to examine the effect of extended destarching
periods, the destarching reaction was allowed to continue as
indicated in Table 2 below. The solids were isolated by filtration,
washed, and dried at 60.degree. C.
2TABLE 2 STARCH REMOVAL MONITORING BY IODINE STAINING units/100 g
corn Reaction Temp % % % % % % Xyl/ Sample Enzyme fiber Time (h)
(.degree. C.) Yield.sup.1 Glu Xyl Gal Ara Man Ara 1 Spezyme
25300.sup.2 0.92 80 86.2 36.2 33.7 6.4 21.2 2.5 1.6 AA-L 2 Spezyme
25300.sup.2 0.92 80 81.9 36.9 33.7 6.2 20.4 2.9 1.6 AA-L 3 Spezyme
25300.sup.2 1.5 80 78.3 37.1 32.9 6.4 21.2 2.4 1.5 AA-L 4 Spezyme
25300.sup.2 3 80 77.5 37.4 32.6 6.5 21.1 2.4 1.5 AA-L Notes for
Table 2: 1 Yield of destarched fiber based upon dry weight of corn
fiber. 2 The activity of thermostable amylase units (TAU) where 1
TAU is defined as the quantity of enzyme that will dextrinize 1 mg
of starch per minute at pH 6.6 and 30.degree. C.
[0235] Table 2 shows that at the indicated concentration of enzyme,
the amount of starch removed increases with increasing reaction
time, but does not reach the anticipated about 68% to about 73%
yield of destarched corn fiber. Further, the % Xyl does not fall
within the expected range (See Example 1), Thus, Table 2
demonstrates that the classical means of determining the endpoint
of starch removal (iodine staining) is not particularly reliable.
In order to more accurately monitor starch removal in the methods
disclosed and claimed herein, the following destarching analysis
method was developed.
Example 3
In Situ Analysis of Destarching Reaction
[0236] The enzymatic degradation of starch in the corn fiber was
monitored in situ by following the appearance of soluble
oligiosaccharides and monosaccharides. The soluble fraction was
measured by infrared spectroscopy using a ReactIR 1000 Spectrometer
(ASI Applied Systems) to measure the peak area at 1050-1010
cm.sup.-1 with baseline definition between 1192-1007 cm.sup.-1.
Enzymatic digestion was assessed as complete when the concentration
of soluble species no longer increased in the destarching reaction
medium. The general details of the destarching were described above
in Example 2. Two different destarching enzymes were utilized. A
summary of the results is shown in FIGS. 1 and 2.
[0237] A number of important features are illustrated by FIGS. 1
and 2. Significantly, the fiber that was not dried prior to
destarching and that contained SO.sub.2 (852 ppm sulfur) as an
artifact of the corn steeping process i.e., wet milled corn fiber,
could be destarched more rapidly than the dried fiber even when
less enzyme was utilized (compare Samples 1 with 4 and Samples 2
with 6). As noted previously, this result was surprising because it
was expected that the SO.sub.2 would act as a biocide, thus
retarding enzymatic degradation of the starch.
[0238] FIG. 1 also illustrates that, with the exception of the
lower concentration of the less active enzyme, all of the
destarching reactions converged at 90% destarching at approximately
20 minutes. Further, prolonged reaction times (about 100 to about
180 min) were required to reach a steady state of concentration of
soluble species.
[0239] FIG. 2 demonstrates that Spezyme AA gave a higher rate of
destarching at equivalent concentration of enzyme and at equivalent
temperature (compare Samples 1 and 2). By increasing the
concentration of the less active Spezyme AA-L, the rate of
destarching could be increased to a rate equivalent to that of
Spezyme AA (compare Samples 2 and 3). Increasing the temperature
from 80.degree. C. to 95.degree. C. at equivalent Spezyme AA
concentration increased the rate of destarching (compare Samples 2
and 5). As previously noted, the use of wet corn fiber containing
SO.sub.2 gave the highest rates of reaction (See Samples 4 and 6.)
These observations illustrate the distinct advantages, e.g., less
enzyme utilization and shorter reaction times, of using wet corn
fiber versus dried corn fiber. Further, these results demonstrate
that destarching can be completed in times significantly shorter
than previously thought. Also, in situ monitoring of reactions by
techniques such as those described in this Example (IR, Raman
spectroscopy) provides the best process for monitoring the
destarching of corn fiber.
Example 4
Simultaneous Destarching and Proteolysis of Corn Fiber at Elevated
pH and Temperature
[0240] Corn fiber (466.7 g, dry wt.) and distilled water (4000 mL)
were added to a 5000 mL 3-necked round-bottomed flask equipped with
an overhead stirrer, a condenser, and a thermometer attached to a
Therm-O-Watch temperature controller. The pH of the mixture was
adjusted to a pH of 8.5 with NaOH. The mixture was heated with
stirring to 80.degree. C. and was held at this temperature with
stirring for approximately 15 minutes. Amylase (GC 521, 16.7 mL,
Genencor, Palo Alto, Calif.) and protease (Protex 6L, 10.0 mL,
Genencor, Palo Alto, Calif.) were added to the mixture
simultaneously. The mixture was stirred for 2.5 hours at 80.degree.
C. with no attempt to control pH. The mixture was filtered through
a 1 mill pore size Buchner funnel. The retained fiber was washed
with eight 2000 mL portions of hot water (45-55.degree. C.)
followed by a single 2000 mL wash with distilled water. The fiber
was then dried at 60.degree. C. in a vacuum oven and submitted for
carbohydrate analysis. The results are summarized in Table 3.
3TABLE 3 NORMALIZED CARBOHYDRATE COMPOSITION OF CORN FIBER TREATED
SIMULTANEOUSLY WITH PROTEASE AND AMYLASE AT AN INITIAL PH OF 8.5 %
% % % % xyl/ Sample Sample Type glu xyl gal ara man arb 1 Untreated
Corn 63.2 18.0 4.5 12.3 1.6 1.5 Fiber 2 Treated Corn Fiber 33.4
36.2 6.4 22.3 1.7 1.6 Notes for Table 3 Sample 1 was used as
received i.e. no destarching and no protease treatment. Sample 2
was destarebed and proteolyzed according to the methods herein.
[0241] This Example demonstrates that simultaneous addition of
amylase and protease at an initial pH of 8.5 allows for rapid and
effective destarching and proteolysis of corn fiber. Further, it is
not necessary to control the pH), which will inherently decrease,
during the reaction. Even further, both enzymes are sufficiently
stable and active at pH 8.5 and 80.degree. C. allowing rapid
destarching and proteolysis of corn fiber.
Example 5
Extraction of Sterol Esters From Corn Fiber
[0242] Three hundred grams of ground corn fiber (3 mm mesh) were
placed in a porous bag and in a soxhlet extractor and subjected to
ethanol extraction for approximately 15 h (hours). The ethanol was
removed in vacuo, thereby providing an ethanol fraction. The
ethanol extracted corn fiber was further extracted with diethyl
ether for approximately 7 h, thereby providing an ether fraction.
Concentration of the ethanol fraction provided 6.7 g of material,
while the ether fraction provided 2.7 g of material.
[0243] Examination of the two fractions by thin layer
chromatography (precoated silica gel 60 F.sub.254 with a layer
thickness of 250 .mu.m; solvent systems: 20:80
MeOH/CH.sub.2Cl.sub.2, 100 CH.sub.2Cl.sub.2, 100 EtOAc) with UV and
phosphomolybidic acid stain detection revealed that only the
ethanol fraction contained an UV visible component (R.sub.f 0.51
(CH.sub.2Cl.sub.2)). This component was thought to be a phytosterol
ferulate ester.
[0244] To confirm this assumption, the ethanol fraction was
partially separated by passing it through a 25.times.4.5 cm column
packed with silica gel using CH.sub.2Cl.sub.2 as the eluting
solvent. After a 200 mL precut, 50 mL fractions were collected. The
fractions containing a UV visible component were pooled and passed
a second time through a 23.times.4.5 cm column packed with silica
gel using 75:25 CH.sub.2Cl.sub.2/hexane as the eluting solvent.
After four 50 mL precut portions, fractions of 10 mL were
collected. The fractions containing an UV visible component, which
appeared essentially free from contaminating material, were pooled
and concentrated to dryness, yielding 168.7 mg of a white amorphous
solid. Examination of this material by field desorption mass
spectroscopy revealed two parent ions {MS (FD) m/e 592 M.sup.+ and
578 M.sup.+}. Accurate mass measurement via electron impact mass
spectroscopy with subsequent elemental composition analysis showed
the following probable molecular formulas.
4TABLE 4A ANALYSIS OF UV VISIBLE COMPONENTS OF CORN FIBER EXTRACT
Compound FD-MS El-MS Calcd Formula Error(ppm) Sitostanol 592
592.4490 592.4491 C.sub.39H.sub.60O.sub.4 0.3 ferulate Campestanol
578 578.4329 578.4335 C.sub.38H.sub.58O.sub.4 1.0 ferulate .sup.1H
(300 MHz, CDCl.sub.3) and .sup.13 C NMR (75 MHz, CDCl.sub.3)
spectroscopy (FIG. 3) confirmed the presence of phytosterol
ferulate.
Example 6
Comparison of Oil Extraction From Starched and Destarched Corn
Fiber
[0245] To compare the yield of the lipid fraction from
non-destarched and destarched corn fiber, 300 g of unground corn
fiber (90% solids, 10% moisture) were extracted with ethanol as
described above in Example 5, thereby providing an ethanol
fraction. Concentration of this ethanol fraction yielded 5.50 g of
oil.
[0246] Three hundred grams of corn fiber were then destarched with
85363 TAU of Spezyme AA for 1.5 h according to the general
procedure described above in Examples 1, 2 and 3. Surprisingly, the
aqueous phase containing the soluble dextrins (corn fiber starch)
had no UV active components (as measured by thin layer
chromatography) at the appropriate R.sub.f. This result indicated
that the phytosterol ferulate esters remained with the destarched
corn fiber. The aqueous phase was extracted four times with heptane
which yielded only 157 mg of oil, thus confirming that very little
of the corn oil is removed from the corn fiber during the
destarching step. The destarched corn fiber was then extracted with
ethanol in a manner identical to that used for the corn fiber that
had not been destarched. After evaporation of the ethanol, 12.18 g
of oil was obtained.
[0247] This Example demonstrates that solvent extraction of corn
fiber yields an oil containing high levels of sitostanol ferulate
(also called stigmastan-3-ol ferulate). This material has been
shown to be effective in lowering human cholesterol. Further, this
Example illustrates that destarching of corn fiber followed by
solvent extraction provides a markedly higher yield of corn fiber
oil ( 12.18 g) relative to that obtained when the corn fiber was
not destarched (5.50 g). Significantly, the yield of corn fiber oil
from unground/corn fiber is much higher than that obtained from
ground corn fiber (9.40 g), even when the ground corn fiber is
subjected to two solvent extractions. Compared to the process of
Moreau et al. (U.S. Pat. No. 5,843,499), which requires grinding of
the corn fiber to a small particle size before extraction with
hexane or supercritical CO.sub.2, extraction of unground and
destarched corn fiber provides a much simpler process, while still
providing higher yields of corn fiber oil. Moreover, the use of wet
corn fiber allows further surprising economies in the industrial
application of the processes herein.
Example 7
Extraction of Oil From Non-Proteolyzed Corn Fiber
[0248] A 20.6 g sample of dry unground corn fiber (not
enzymatically treated) was extracted with 250 mL of hexane for 60
min. The liquid was then filtered from the fiber through a 10-15
.mu.m glass fitted funnel, washing with enough hexane so as to
recover a total of 250 mL of solvent. The fiber was then removed
from the frit and it was rinsed with 50 mL of hexane. The hexane
was filtered through filter paper prior to concentration in vacuo.
The procedure was repeated 6 times giving an average yield of
0.2878.+-.0.0254 g. (1.40.+-.0.12 wt. % oil). The same lot of corn
fiber with a 60% moisture content was also extracted for 75 minutes
in similar fashion with 219 mL of ethyl acetate or isopropanol at
the indicated temperature. Treated corn fiber refers to corn fiber
that has been treated to amylase and protease steps as has been
described previously. The treated samples were run twice and the
average value is given.+-.the standard deviation.
5TABLE 4B COMPARISON OF CORN FIBER OIL EXTRACTION FROM PROTEOLYZED
AND NON-PROTEOLYZED CORN FIBER Corn Fiber Temperature Solvent Type
Oil Mass Oil Wt. % 22.degree. C. i-PrOH Untreated 0.3725 1.81
22.degree. C. EtOAc Untreated 0.0.3574 1.73 22.degree. C. i-PrOH
Treated 0.8184 .+-. 0.0031 3.97 .+-. 0.015 66.degree. C. i-PrOH
Untreated 0.7504 3.64 66.degree. C. EtOAc Untreated 0.6867 3.33
66.degree. C. i-PrOH Treated 1.3718 .+-. 0.0442 6.66 .+-. 0.21
Notes for Table 4B: Untreated corn Fiber was used as received i.e.,
not destarched or proteolzyed. Treated corn Fiber was destarched
and proteolyzed.
[0249] It is surprising that compared to the process of Moreau et
al. (U.S. Pat. No. 5,843,499), a 4.5 fold increase is seen (from
0.31 wt. %) in the amount of extractable oil from corn fiber.
Furthermore, the process of Moreau et al. requires that the fiber
be dried and ground prior to extraction. These steps have
significant associated energy costs associated which adversely
affects the economics of the process, especially if carried out on
an industrial scale.
[0250] It has been determined that the drying and grinding steps
are unnecessary to obtain useful amounts of extractable oil. By
extracting wet fiber at elevated temperature the extraction
efficiency has been found to be nearly double what can be obtained
at room temperature or obtained from dry, unground fiber extracted
with hexane. Furthermore, amylase and protease treatment allows
further increases in the amount of extractable oils, nearly double
in this Example on a same weight basis.
Example 8
Effect of Caustic Extraction Conditions on the Yield and
Concentration of Corn Fiber Components
[0251] The following procedure was used to examine caustic
extraction of corn fiber, specifically the influence of NaOH
concentration, temperature, time, and method of arabinoxylan
precipitation on cellulose and arabinoxylan yield and
composition.
[0252] Destarched corn fiber was added to a 500 mL three-neck glass
round bottom flask containing a solution of NaOH at 80.degree. C.
The flask was equipped with a mechanical stirrer and reflux
condenser. The reaction was stirred for the designated time and
temperature before cooling to room temperature. The mixture of
destarched corn fiber in NaOH was then filtered at room temperature
to remove the cellulose material. The cellulose material was washed
twice with water. The alkaline filtrate was collected to provide a
liquid comprising arabinoxylan. The cellulose material was further
treated by soxhlet extraction with water for about 14 hours. After
extraction, the cellulose material was dried at 60.degree. C. under
house vacuum. The alkaline filtrates were combined and the pH of
the combined filtrate was adjusted to 5, followed by filtration
through celite. Alcohol (about 3/1 ROH/filtrate liquids) was added
to the filter cake, giving a gummy precipitate containing
arabinoxylan. The precipitate was filtered to remove the alcohol,
thereby providing an arabinoxylan-containing precipitate. When
nearly all of the liquid had been removed from the precipitate,
fresh alcohol was added to harden the arabinoxylan precipitate. The
isolated arabinoxylan precipitate was dried at 60.degree. C. under
vacuum.
[0253] In each case, precipitation of the arabinoxylan with alcohol
gave a highly colored (tan to black) solid containing arabinoxylan.
For each case, the arabinoxylan was redissolved in water (about. 10
wt. % solids) and precipitated with acetic acid (ca. 4/1
HOAc/water) which provided a white solid.
6TABLE 5 EFFECT OF EXTRACTION CONDITIONS ON ARABINOXYLAN
(PROPERTIES) % % Total.sup.(5) Total.sup.(5) % % % % NaOH Temp
Yield Yield Carb Carb Glu Xyl Gal Ara Sample (M) (.degree. C.) ROH
HOAc ROH HOAc ROH ROH ROH ROH 1.sup.(1) 0.63 101 40.3.sup.(3) 18.7
80.8 88.7 1 56 10 34 2.sup.(1) 0.63 101 38.4.sup.(3) 26.7 78.1 86.4
1 54 10 35 3.sup.(1) 1.25 100 43.1.sup.(3) 29.6 76.6 88.9 3 56 9 32
4.sup.(1) 1.25 102 40.2.sup.(3) 24.1 83.0 89.7 1 56 9 34 5.sup.(2)
2.5 RT 31.1.sup.(3) 22.7 78.6 84.0 7 51 9 33 6.sup.(1) 2.5 60
47.0.sup.(3) 35.2 73.4 85.8 8 51 10 31 7.sup.(1) 2.5 80
72.4.sup.(4) Nd 42.4 nd 7 50 9 34 8.sup.(2) 2.5 80 60.0.sup.(4)
32.7 71.7 85.9 8 51 9 32 9.sup.(2) 2.5 80 58.7.sup.(4) 34.4 72.1
86.2 7 51 9 32 10.sup.(2) 2.5 80 59.9.sup.(4) 32.9 66.6 82.6 7 50 9
35 11.sup.(2) 2.5 102 45.3.sup.(3) 30.4 71.9 84.2 5 54 10 31 12 2.5
120 50.9.sup.(3) 37.1 80.6 88.0 3 55 9 33 13.sup.(1) 3.75 101
52.6.sup.(3) 13.5 69.4 87.8 5 54 10 32 % Xyl/ % % % % % Xyl/ Man
Ara Glu Xyl Gal Ara Man Ara % % Sample ROH ROH Ac Ac Ac Ac Ac Ac
C.sup.(6) H.sup.(6) 1.sup.(1) 0 1.7 1 54 10 35 0 1.5 42.3 6.7
2.sup.(1) 0 1.5 1 54 10 35 0 1.5 41.9 7.0 3.sup.(1) 0 1.7 3 54 10
34 0 1.6 42.1 7.1 4.sup.(1) 0 1.7 1 55 9 35 0 1.6 42.3 6.6
5.sup.(2) 0 1.6 7 51 8 34 0 1.5 36.7 5.4 6.sup.(1) 0 1.7 9 51 9 31
0 1.6 31.4 4.6 7.sup.(1) 0 1.5 nd nd nd nd nd nd 15.7 2.4 8.sup.(2)
0 1.6 7 51 9 32 0 1.6 35.0 6.0 9.sup.(2) 0 1.6 7 52 9 32 0 1.6 15.3
2.3 10.sup.(2) 0 1.4 6 51 9 33 0 1.5 28.1 4.9 11.sup.(2) 0 1.7 8 51
9 32 0 1.6 37.9 5.9 12 0 1.7 4 55 9 32 0 1.7 38.0 6.7 13.sup.(1) 0
1.7 5 53 9 33 0 1.6 36.9 6.1
[0254]
7TABLE 6 EFFECT OF EXTRACTION CONDITIONS ON PROPERTIES OF CELLULOSE
MATERIAL Total % Total % Yield Yield % % % % % % Xyl/ Polysac-
Polysac- NaOH Time Temp Yield Total.sup.(5) Glu Xyl Gal Ara Man Ara
% % charide charide % Sample (M) (h) (.degree. C.) Cellulose Carb
ROH ROH ROH ROH ROH ROH C.sup.(6) H.sup.(6) (ROH) (HOAc) Cellulose
I 1.sup.(1) 0.63 1 101 28.8 82.8 63 21 4 10 2 2 45.4 7.4 69.1 47.5
Nd 2.sup.(1) 0.63 8 101 24.5 90.4 68 17 3 8 3 2 42.6 6.9 62.9 51.2
Nd 3.sup.(1) 1.25 1 100 25.4 83.9 71 15 3 7 3 2 43.0 7.0 68.5 55
100 4.sup.(1) 1.25 8 102 22.0 80.1 72 15 3 7 3 2 42.4 6.7 62.2 46.1
Nd 5.sup.(2) 2.5 4 RT 41.8 69.9 55 24 6 13 9 2 45.5 7.2 72.9 64.5
100 6.sup.(1) 2.5 4 60 30.0 76.6 82 8 2 5 3 2 25.2 3.6 77.0 65.2 50
7.sup.(1) 2.5 0.5 80 20.7 79.1 83 8 2 5 2 2 41.0 6.5 93.1 nd 75
8.sup.(2) 2.5 1 80 21.5 78.6 84 7 2 4 3 2 35.7 5.8 82.1 54.2 50
9.sup.(2) 2.5 2 80 21.3 77.9 87 6 1 3 3 2 36.9 6.0 80.0 55.7 nd
10.sup.(2) 2.5 4 80 22.9 71.1 82 8 2 5 3 2 36.1 5.3 82.8 55.8 50
11.sup.(2) 2.5 4 102 17.4 82.4 91 4 1 2 3 2 39.1 6.2 62.7 47.8 12
2.5 4 120 15.2 92.0 91 4 1 1 3 3 43.2 6.9 66.1 52.3 nd 13.sup.(1)
3.75 8 101 22.9 60.4 77 11 2 7 3 2 37.2 5.8 75.5 36.4 nd Notes for
Tables 5 and 6: All reactions at 5% solids. nd = not determined.
.sup.(1)Bacillus was used for destarching fiber. .sup.(2)A. Oryzac
was used for destarching corn fiber. .sup.(3)ROH = MeOH.
.sup.(4)ROH = EtOH. .sup.(5)Carbohydrate balance from carbohydrate
analysis. .sup.(6)Carbon and hydrogen analysis was performed on the
arabinoxylan samples isolated by ROH precipitation.
[0255] For an arabinoxylan consisting of 90% C5 sugars and 10% C6
sugars, the expected values are 45.39% carbon and 6.11% hydrogen.
For cellulose material, the expected values are 44.45% carbon and
6.21% hydrogen.
[0256] A number of significant results are demonstrated by Tables 5
and 6. First, the method of arabinoxylan precipitation from the
caustic solution has a significant impact on the purity and quality
of the arabinoxylan obtained. Precipitation of the arabinoxylan
from the caustic extraction solution using EtOH generally resulted
in higher yields (59 to 72%, Samples 7 to 10). However, analysis by
HPLC indicated that only 42 to 72% of the samples comprised
carbohydrate; elemental analysis revealed that the carbon and
hydrogen percentages significantly deviated from the theoretical
values. Furthermore, the samples precipitated using EtOH were
highly colored (brown-black) indicating significant contamination
by salts and other organic materials. Precipitation of the
arabinoxylan from the caustic extraction solution using MeOH
resulted in lower yields of arabinoxylan (31 to 52%, Samples 1 to
6, 11 to 13), improved carbohydrate balance (72 to 80%), and
slightly improved color (brown). Precipitation of the arabinoxylan
from the caustic extraction solution using HOAc gave a more pure
form of arabinoxylan with yields in the range of 24 to 37%, good
carbohydrate balance (83 to 90%), and excellent color (white-off
white). These results indicate that precipitation with HOAc
provides arabinoxylan of higher purity and significantly improved
color.
[0257] Second, the best combination of maximum arabinoxylan yield
and cellulose purity, based on a single caustic extraction, was
obtained at 2.5 M NaOH concentration in the temperature range of
60.degree. C. to 120.degree. C. Under these conditions, the percent
xylose (as measured by carbohydrate analysis) is reduced slightly
from that observed under the other stated conditions i.e., from 54
to 55% to 51 to 52%, but the xyl/arb ratio remains constant at
about 1.6.
[0258] With respect to the cellulose component, the highest yield
of derivatizable cellulose was obtained at a NaOH concentration of
2.5 M at 25.degree. C. As the temperature or reaction time is
increased at a fixed concentration of 2.5 M NaOH, the yield of
cellulose decreases but, very importantly, the .alpha.-purity (%
glucose) of the cellulose increases. The highest level of purity
for the cellulose (91%) was obtained at 2.5 M NaOH at 100.degree.
C. and 120.degree. C. It should be noted that the percentage of
cellulose I in these samples was unpredictable and ranged from 50
to 100%.
[0259] Collectively, Example 8 demonstrates that, for a single
extraction, the best balance of conditions for optimal
polysaccharide yield and purity is 2.5 M NaOH at 80.degree. C.
followed by precipitation of the extracted arabinoxylan in acetic
acid.
Example 9
Examination of Different Caustic Types on Extraction
[0260] The general procedure of Example 8 was used to examine the
effect of different caustic types, i.e., KOH and Ca(OH).sub.2, on
the separation of destarched corn fiber into arabinoxylan and
cellulose fractions in comparison to when the caustic was NaOH.
8TABLE 7 EFFECT OF CAUSTIC TYPE, TEMPERATURE AND CONCENTRATION ON
ARABINOXYLAN EXTRACTION % % Total.sup.(5) Total.sup.(5) % % % NaOH
Time Temp Yield Yield Carb Carb Glu Xyl Gal Sample (M) (h)
(.degree. C.) ROH HOAC ROH HOAc ROH ROH ROH 1.sup.(1) 1.78 4 102
38.4.sup.(3) 31.2 83.1 83.3 1 53 10 KOH 2.sup.(1) Sat. 4 60
18.8.sup.(3) 15.2 48.4 na 0 54 9 Ca(OH).sub.2 3.sup.(1) Sat. 4 102
30.7.sup.(3) 22.2 85.7 87.8 0 55 10 Ca(OH).sub.2 4.sup.(1) Sat. 4
120 26.5.sup.(3) 12.8 81.4 84.7 1 55 10 Ca(OH).sub.2 5.sup.(1) Sat.
8 100 28.4.sup.(3) 22.8 79.2 85.7 0 55 10 Ca(OH).sub.2 6.sup.(1)
2.5 4 60 47.0.sup.(3) 35.2 73.4 85.8 8 51 10 NAOH 7.sup.(2) 2.5 4
102 453.sup.(3) 30.4 71.9 84.2 5 54 10 Ca(OH).sub.2 8 2.5 4 120
50.9.sup.(3) 37.1 80.6 88.0 3 55 9 Ca(OH).sub.2 % % Xyl/ % % % % %
Xyl/ Arabin Man Ara Glu Xyl Gal Ara Man Ara % % Sample oxylan ROH
ROH ROH Ac Ac Ac Ac Ac Ac C.sup.(6) H.sup.(6) 1.sup.(1) 36 0 1.5 1
54 10 36 0 1.5 41.68 7.02 2.sup.(1) 37 0 1.4 0 55 9 36 0 1.5 41.03
6.16 3.sup.(1) 35 0 1.5 0 55 9 35 0 1.6 42.86 7.11 4.sup.(1) 34 0
1.6 0 55 9 35 0 1.6 42.04 7.43 5.sup.(1) 35 0 1.6 0 55 9 36 0 1.5
42.47 7.08 6.sup.(1) 31 0 1.7 9 51 9 31 0 1.6 31.4 4.6 7.sup.(2) 31
0 1.7 8 51 9 32 0 1.6 37.9 5.9 8 33 0 1.7 4 55 9 32 0 1.7 38.0
6.7
[0261]
9TABLE 8 EFFECT OF CAUSTIC TYPE, TEMPERATURE AND CONCENTRATION ON
CELLULOSE % % % % % % Xyl/ NaOH Time Temp Yield Total.sup.(5) Glu
Xyl Gal Ara Man Ara % % Entry (M) (h) (.degree. C.) Cellulose Carb
ROH ROH ROH ROH ROH ROH C.sup.(6) H.sup.(6) 1.sup.(1) 1.78 4 102
25.8 95.0 68 17 3 8 3 2.0 44.72 7.18 KOH 2.sup.(1) Sat. 4 60
>100 14.1 51 26 5 16 2 1.6 14.26 3.57 Ca(OH).sub.2 3.sup.(1)
Sat. 4 102 >100 22.7 55 21 5 13 7 1.6 13.06 3.57 Ca(OH).sub.2
4.sup.(1) Sat. 4 120 >100 16.4 62 22 4 12 0 1.8 9.51 3.04
Ca(OH).sub.2 5.sup.(1) Sat. 8 100 >100 11.2 57 23 4 14 2 1.7
12.55 2.19 Ca(OH).sub.2 6.sup.(1) 2.5 4 60 30.0 76.6 82 8 2 5 3 2
25.2 3.6 NaOH 7.sup.(2) 2.5 4 102 17.4 82.4 91 4 1 2 3 2 39.1 6.2
NaOH 8 2.5 4 120 15.2 92.0 91 4 1 1 3 3 43.2 6.9 NaOH
[0262] Carbon and hydrogen analyses were performed on the
arabinoxylan samples isolated by ROH precipitation.
[0263] For an arabinoxylan consisting of 90% C5 sugars and 10% C6
sugars, the expected vales are 45.39% carbon and 6.11% hydrogen.
For cellulose, the expected values are 44.45% carbon and 6.21%
hydrogen.
[0264] From Table 7 it is apparent that caustic extraction of the
corn fiber with either KOH or Ca(OH).sub.2 provided a lower yield
of arabinoxylan relative to that obtained when NaOH was utilized as
the base. Also, the arabinoxylan obtained using either KOH or
Ca(OH).sub.2 had a lower percent glucose and slightly higher
percent arabinose (as measured by carbohydrate analysis).
[0265] From Table 8 it is apparent that, in terms of the cellulose
component, when Ca(OH).sub.2 was utilized as the base, the
excessively high yields and the poor carbohydrate balance indicate
that Ca(OH).sub.2 is not removed from the cellulose fraction,
probably due to the low solubility of Ca(OH).sub.2 in water. The
carbohydrate and the carbon, hydrogen analyses indicated that the K
salts were most easily removed from the cellulose relative to
Ca(OH).sub.2 and NaOH. Importantly, the .alpha.-purity (% glucose)
of the cellulose obtained using Ca(OH).sub.2 and KOH (51 to 68%)
was much less than that obtained using NaOH (82 to 91%).
[0266] When taken together, the results from Tables 7 and 8
indicate that NaOH provides the most satisfactory balance of
qualities for both the arabinoxylan and cellulose aspects of the
present invention. KOH also provides satisfactory results, but it
is not as good as NaOH.
Example 10
Examination of Solids Level on NAOH Extraction
[0267] Following the general procedure of Example 8 above,
destarched corn fiber was separated into arabinoxylan and cellulose
fractions by treating the corn fiber with NaOH at 80.degree. C. for
1 hour and varying the solids and NaOH concentration as specified
in the following tables.
10TABLE 9 EFFECT OF SOLIDS LEVEL AND NAOH CONCENTRATION ON
ARABINOXYLAN PROPERTIES NaOH % % Yield Total % % % % % Xyl/ Sample
(M) Solids Ara Carb Glu Xyl Ara Man Ara % C % H 1 2.5 5 32.7 85.9 7
51 9 32 0 1.6 34.98 6.02 2 2.5 10 31.1 81.9 6 51 9 34 0 1.5 34.7
6.35 3 2.5 15 32.0 89.3 4 51 11 34 0 1.5 39.58 6.04 4 2.5 20 27.5
75.2 2 53 9 35 0 1.5 40.08 7.19 5 5 10 33.3 85.7 8 50 9 33 0 1.5
33.72 5.41 6 10 20 33.9 85.0 9 50 9 32 0 1.6 29.53 6.62
[0268]
11TABLE 10 EFFECT OF SOLIDS LEVEL AND NAOH CONCENTRATION ON
CELLULOSE PROPERTIES NaOH % % Yield Total % % % % % Xyl/ Sample (M)
Solids Cellulose Carb Glu Xyl Gal Ara Man Ara % C % H 1 2.5 5 21.5
78.6 84 7 2 4 3 1.8 35.73 5.75 2 2.5 10 23.0 79.5 79 10 2 5 3 1.9
36.15 5.96 3 2.5 15 Nd 64.2 63 20 4 11 2 1.7 34.37 5.16 4 2.5 20
26.4 84.2 71 16 3 8 2 2.0 39.19 6.15 5 5 10 25.9 58.3 81 9 2 5 3
1.6 35.37 5.68 6 10 20 24.8 70.3 85 7 2 3 3 2.1 32.49 5.33
[0269] It was found that at above 10% solids i.e., the amount of
corn fiber utilized in the caustic extraction step, the viscosity
of the caustic solution/corn fiber mixture at room temperature was
high, making it difficult to separate the solution comprising
arabinoxylan from the cellulose material. The amount of solids in
the reaction mixture did not have a significant effect on
arabinoxylan yield or composition. However, surprisingly,
increasing the solids at a constant NaOH concentration led to lower
.alpha. purity (% glucose) for the cellulose. By increasing the
NaOH concentration, the purity of the cellulose could be increased
significantly so that, for example, at 20% solids and 10 M NaOH,
the purity of the cellulose was comparable to that obtained at 2.5
M NaOH and 5% solids.
Example 11
Effect of Differing Caustic Strength, Times and Temperatures on
Arabinoxylan Extraction
[0270] Examples 9 and 10 above provide results when both
polysaccharide fractions (cellulose material and arabinoxylan) are
obtained as products when the objective was obtaining the maximum
purity for each material. However, with the invention herein it is
possible to achieve a suitable industrial process by focusing on
one polysaccharide component.
[0271] In this regard, the rate of extraction of the arabinoxylan
fraction was examined by using a ReactIR 1000 Spectrometer as
described above in Example 3. The extraction of the arabinoxylan
from the corn fiber was monitored by following the appearance of
solubilized arabinoxylan in solution. The soluble fraction was
measured by infrared spectroscopy by measuring the peak area at
1050-1010 cm.sup.-1 with baseline definition between 1098-988
cm.sup.-1. The extraction of arabinoxylan from the corn fiber was
considered to be complete when the concentration of soluble species
in the extractant no longer increased.
[0272] The results are summarized in FIG. 4, in which a number of
key points are illustrated. First, the extraction of arabinoxylan
from the corn fiber can occur very rapidly depending upon the
combination of temperature and caustic strength utilized.
Surprisingly, the extraction rate is quite rapid at low caustic
strengths, provided the temperature is high. (See FIG. 4, 0.75 M
NaOH, 90.degree. C.) That is, temperature appears to have a more
significant effect on arabinoxylan extraction rates than caustic
strength alone. Conversely, a lower temperature can be used when
the caustic strength is high. (See 1.25 M NaOH, 70.degree. C.) In
the case of 1.25 M NaOH at 90.degree. C. (higher caustic strength
and higher temperature), essentially all of the arabinoxylan is
extracted from the corn fiber in about 20 minutes and approximately
90% of arabinoxylan is extracted in about 8 minutes. These
unexpected observations illustrate that significant quantities of
the arabinoxylan can be extracted from the corn fiber in much
shorter times at lower caustic strengths than previously disclosed.
This indicates that arabinoxylan can be extracted from corn fiber
using a continuous extraction process which greatly simplifies the
isolation of arabinoxylan from corn fiber and allows marked
economies in industrial processes.
[0273] Furthermore, in contrast to prior reports, no ferulate
esters were observed even under the more mild extraction
conditions. (L. Saulnier, C. Marot, E. Chanliaud, J. -F. Thibault,
Carbohydrate Polym., 1995, 26, 279-287.)
Example 12
Extraction of Arabinoxylan From Corn Fiber at Low Caustic Strength
and Short Contact Times
[0274] Dried, destarched corn fiber (24.5 g) was rehydrated with
200 mL H.sub.2O and heated to 65.degree. C. Hot, 0.5 M aqueous NaOH
(225 ml) was added and the resulting mixture (70 to 75.degree. C.)
was stirred for 7 minutes before filtering through a Buchner
funnel. The residual solids were washed with water (400 mL) for 8
minutes. The extractant contained approximately 2 to 3% solids and
was centrifuged for 10 min at 2000 rpm, yielding a paste and a
liquid. The liquid was decanted from the paste and concentrated.
Arabinoxylan was precipitated from the liquid using AcOH. 3.71 g of
arabinoxylan (15% yield from destarched corn fiber) was
obtained.
[0275] This Example, which is illustrative of contacting the corn
fiber very briefly with caustic, illustrates that significant
yields of arabinoxylan can be obtained very rapidly with little or
no effect on the ability to filter the caustic from the corn
fiber.
Example 13
Effect of Multiple Caustic Contacting Steps on Arabinoxylan
Extraction
[0276] Dried, destarched corn fiber (25.0 g) was rehydrated with
200 mL H.sub.2O and heated to 65.degree. C. The corn fiber was
added to a 600 mL glass fritted funnel with a pore size of 70 to
100. Warm 0.5 M aqueous NaOH (100 mL) was added and the resulting
mixture (55 to 60.degree. C.) was stirred for 3 seconds before
filtering. This process was repeated 7 times (19 min total) and an
additional time with 200 mL of water (no caustic). The filtrate
from each caustic extraction was collected, concentrated and
treated with AcOH to precipitate arabinoxylan. The overall yield of
arabinoxylan from the combined caustic extractions was 13.3%. FIG.
5 shows the isolated yield, output concentration and total yield
plotted for each extraction.
[0277] This Example demonstrates that arabinoxylan can be extracted
from corn fiber in a continuous process by using multiple short
contact steps of caustic with destarched corn fiber. It is
significant in this Example that very low caustic strength can be
used to obtain satisfactory extraction of arabinoxylan from the
corn fiber. In contrast to prior reports, no ferulate esters were
observed by NMR spectroscopy. (L. Saulnier, C. Marot, E. Chanliaud,
J. -F. Thibault. Carbohydrate Polym., 26(1995), pp. 279-287.
Example 14
Microscopic Examination of Reason for Filtering Difficulty
[0278] Long exposure of destarched corn fiber to caustic was seen
to cause difficulty in separating the caustic solution containing
arabinoxylan from the cellulose material. In order to determine the
source of the difficult filtration, the heterogeneous caustic
extraction mixture was examined by optical microscopy.
[0279] After caustic treatment and treatment with AcOH, the
cellulose material was made of fibers of significant length. In the
absence of other contaminates, filtration of these fibers would not
be problematic. However, it was found that the presence of lipid,
proteinacious material, and/or starch granules in the heterogeneous
caustic extraction mixture caused difficulty in filtering the
cellulose fiber from the caustic extractant. (FIG. 6.) This
difficulty appeared to be caused by a clogging of the filter with
the small particle size proteinacious material and/or starch
granules. Accordingly, experiments were performed to determine ways
in which the filtration process could be optimized.
Example 15
Effect of Proteolysis and Lipid Extraction on Corn Fiber Purity and
Ease of Filtration of the Liquid Comprising Arabinoxylan
[0280] This Example demonstrates the effect of proteolysis and
lipid extraction on such variables as corn fiber purity and
filtration ease. This Example further demonstrates that the
combination of destarching and proteolysis of corn fiber provides a
significant higher yield of corn fiber oil relative to that
obtained from unground or ground corn fiber that was not subjected
to these enzymatic reactions.
[0281] A 100 mL round-bottom flask was charged with 5 g (3.67 g dry
weight basis) of destarched corn fiber (Sample 1), 72 mL of
deionized water, and 8.0 mL of 1.0 M sodium phosphate buffer (pH
7). The mixture was heated to 40.degree. C. before addition of 1.0
mL (42 GSU) of Purafect 4000L, a protease enzyme (Genencor, Palo
Alto, Calif.). The mixture was stirred for 18 h at 40.degree. C.
before being allowed to cool to room temperature over a 30 min time
period. The sample was filtered, washed with 250 mL of deionized
water, and dried at 50.degree. C. in vacuo to provide 2.7 g of
coarse fiber (Sample 2). Fine particles were removed from the
filtrate by centrifugation. The fines were re-suspended in water,
removed from the water by centrifugation, and dried at 50.degree.
C. in vacuo which gave 0.28 g of fines (Sample 3). The filtrate
from which the fine particle solids were removed was concentrated
to dryness which gave a yellow solid (Sample 4). The results are
summarized in Table 11.
12TABLE 11 EFFECT OF DESTARCHING AND PROTEOLYSIS ON CORN FIBER
FILTRATION Sam- % % % % % % Xyl/ ple Description nitrogen Glu Xyl
Gal Ara Man Ara 1 Destarched 1.26 33.7 35.5 5.3 23.7 2.0 1.5 Corn
Fiber 2 Destarched, 0.19 32.9 38.1 5.7 22.0 1.5 1.7 Proteolyzed
Corn Fiber 3 Fines From 1.09 60.6 18.5 4.8 10.9 5.2 1.7 Proteolyzed
Corn Fiber Filtrate 4 Yellow 1.62 nd nd nd nd nd nd Solid
[0282] This Example illustrates that treatment of destarched corn
fiber with a protease enzyme significantly lowers the percent
nitrogen in the remaining coarse corn fiber to near the limit of
detection (compare Samples 1 and 2). Significantly, nearly all of
the protein is found in the fines and filtrate liquids (Samples 2
and 4.) Furthermore, proteolysis of the destarched corn fiber
lowers the percent glucose while increasing the percent xylose
(Sample 2). In contrast, the unwanted fines have significantly more
glucose (60.6%, Sample 3).
[0283] Collectively, this data demonstrates that treatment of
destarched corn fiber with a protease enzyme removes nearly all of
the protein fraction, thus providing a cleaner corn fiber that
ultimately leads to a cleaner arabinoxylan and cellulose fraction
(vide infra).
[0284] In a further examination of the filtration step, dried,
destarched, protease treated, and oil extracted corn fiber (19.3 g)
was rehydrated with H.sub.2O (100 mL) at 60.degree. C. for 30
minutes. To this was added hot (95.degree. C.) 1.0 M aqueous NaOH
(250 mL) and the resulting mixture (80.degree. C.) was stirred and
heated to 95.degree. C. over 20 minutes. The slurry had a volume of
approximately 400 mL and was filtered in a 600 mL glass fritted
funnel with a pore size of 70-100 .mu.m. Filtration of this
destarched, proteolyzed corn fiber was completed in only 2 minutes
with subsequent H.sub.2O washes (.about.250 mL each) taking between
30-45 seconds. The filtrate was concentrated before precipitation
of the arabinoxylan with HOAc to provide 6.30 g of hemicellulose
(33% yield). Significantly and surprisingly, without removal of the
protein fraction, such filtration would normally require about 12
h. Thus, in a preferred method of the invention herein, it is
critical to subject the corn fiber, whether destarched or not, to a
proteolysis treatment.
[0285] A similar reaction was carried out utilizing destarched and
protease treated corn fiber from which the corn fiber oil had not
been extracted. This corn fiber was treated to more extreme caustic
conditions (2.5 M NaOH at 80.degree. C. for 4 hours). Again,
significantly and surprisingly, filtration and rinsing also
occurred rapidly. This demonstrates that proteolysis of the corn
fiber also increases the filtration efficacy even when corn fiber
oil has not been extracted from the corn fiber.
[0286] In summary, this Example demonstrates that after removal of
the protein component from the corn fiber, separation of the
caustic slurry from the cellulose fraction by filtration can be
carried out without difficulty. This method is very efficient and
suitable for an industrial setting. It is not necessary to carry
out partial fractionation of the corn fiber to achieve more
reasonable filtration speeds as described earlier. Short caustic
treatments as well as long, high caustic strength and high
temperature treatments both provide reaction mixtures that can be
filtered with ease. Finally, it is not necessary to remove the oil
content from the corn fiber prior to caustic extraction to achieve
rapid filtration.
Example 16
Examination of One-Step Destarching and Proteolysis of Corn
Fiber
[0287] A 5000-mL 3-necked round-bottomed flask, equipped with an
overhead stirrer, an air-cooled condenser, and a thermometer
connected to a Therm-o-watch temperature controller, was charged
with approximately 1082.9 g of wet corn fiber (385.7 g dry wt.),
3150 mL of water and 270 mL of 1 M sodium phosphate buffer, as pH
7. The mixture was heated to approximately 80.degree. C. at which
point approximately 15 mL of Spezyme AA was added and the mixture
was stirred at 80.degree. C. for about 2.5 hours. The mixture was
allowed to cool to 40.degree. C. and about 25 mL of Purafect 4000L
was added and the mixture was stirred for approximately 11 hours.
The mixture was filtered through a Buchner funnel and washed with
9.times.1000 mL of hot (about 60.degree. C.) water, followed by
3.times.1000 mL of deionized water to assure effective removal of
corn fiber fines. The resulting material was dried at 50.degree. C.
in a vacuum oven to produce 204 g of proteolyzed, destarched corn
fiber at approximately 53% recovery from the starting corn
fiber.
[0288] This Example illustrates that the amylase and protease
treatment can occur sequentially in the same reaction vessel with
very effective removal of the starch and protein fractions.
Example 17
Extraction of Destarched, Proteolyzed Corn Fiber Providing a Novel
Corn Fiber Lipid Fraction
[0289] A 20.6 g sample of dry untreated corn fiber and a 20.6 g
sample of destarched, proteolyzed corn fiber was extracted with 250
mL of hexane for 60 minutes. The liquid was then filtered from the
fiber through a 10-15 .mu.m glass flitted funnel, washing with
enough hexane so that a total of 250 mL of liquids were recovered.
The fiber was then rinsed with an additional 50 mL of hexane. The
hexane extract was filtered through filter paper and concentrated
in vacuo. The recovered oil samples were then subjected to HPLC
analysis according to the method of Moreau et al. (U.S. Pat. No.
5,843,499). Each experiment was repeated three times. Typical
results are shown in FIG. 7.
[0290] Quite surprisingly and unexpectedly, the composition of the
corn fiber lipid fraction from the proteolyzed, destarched corn
fiber was significantly different from that observed from untreated
corn fiber. Specifically, the concentration of phytosterols in the
lipid fraction of the destarched, proteolyzed corn fiber was
2.1.+-.0.5 times greater than the concentration of phytosterols in
the lipid fraction of nontreated corn fiber. Furthermore, the
concentration of phytosterol ferulates in the lipid fraction of the
destarched, proteolyzed corn fiber was 1.9.+-.0.25 times less than
the concentration of phytosterol ferulates in the lipid fraction of
nontreated corn fiber. That is, destarching and proteolysis of corn
fiber provides for corn fiber oil more enriched in phytosterols
with less phytosterol ferulate relative to corn fiber oil from
untreated fiber.
Example 18
Effect of Varying Parameters on Extraction of Corn Fiber Oil
[0291] Corn fiber (20.6 g) that had been subjected to different
types of treatment (vide infra), was exposed to 206 mL of an
organic solvent with stirring at room temperature for 1 hour. The
solution was then filtered, concentrated under reduced pressure,
and any residual solvent was removed under high vacuum (0.05-0.1
torr) thereby providing a corn fiber oil. The amount of oil that
was isolated and the analyses thereof are summarized in Table 12,
below. Sample 3 is a comparative example reflecting the art
disclosed in U.S. Pat. No. 5,843,499, to Moreau et al. Phytosterol
ferulate esters content was analyzed by HPLC utilizing the method
disclosed in that patent.
13TABLE 12 ANALYSIS OF EXTRACTION CONDITIONS ON CORN FIBER OIL
Campestanyl Sitostanyl Extractable oil phytosterol Special ferulate
ferulate Ferulate Oil extracted from fiber ferulate Sample Solvent
Conditions (wt. % of oil) (wt. % of oil) esters (mg) (mg) (wt. %)
(mg)/fiber (g) 1 Hexane U, D, P1 1.58 3.28 37.5 772.4 3.75 1.823 2
Hexane G, D, P1 0.70 1.44 18.5 866.6 4.21 0.901 3 Hexane G, D, R
1.54 3.03 17.2 375.9 1.82 0.832 4 Hexane G, D, A 0.69 1.42 11.5
543.8 2.64 0.557 5 Hexane U, D, R 1.59 3.13 5.4 113.7 0.55 0.260 6
EtOAc U, W, P2 1.27 2.47 24.1 643.4 3.12 1.167 7 Acetone U, D, P2
1.62 2.91 22.6 499.3 2.42 1.096 8 EtOAc U, D, P2 1.34 2.51 20.6
533.8 2.59 0.997 9 MEK U, D, P2 1.37 2.47 19.6 509.9 2.48 0.952 10
Methanol U, D, P2 1.48 2.73 17.5 415.1 2.02 0.850 11 Heptane U, D,
P2 1.29 2.39 17.2 467.2 2.27 0.835 12 Hexane U, D, P2 1.13 2.23
15.6 464.6 2.26 0.759 13 Isopropanol U, D, P2 1.28 2.43 14.2 382.5
1.86 0.690 14 Hexane U, W, P2 1.21 2.26 9.4 270.7 1.31 0.455 Notes
for Table 12: U = unground, G = Ground, R = Non-enzymatically
treated fiber, A = amylase treated, P1 = amylase & protease (18
h) treated, P2 = amylase & protease (11 h) treated, D = Dry
fiber, W = Wet fiber (65% moisture). NOTES FOR TABLES 7 AND 8: All
reactions at 5% solids. nd = not determined. (1) Bacillus was used
for destarching corn fiber. (2) A. Oryzae was used for destarching
corn fiber. (3) ROH = MeOH (4) ROH = EtOH (5) Carbohydrate balance
from carbohydrate analysis.
[0292] As noted, Sample 3 is a comparative example utilizing the
method disclosed in U.S. Pat. No. 5,843,499 (dry, ground fiber,
hexane extraction). Comparison of Samples 3 and 5 (dry, unground
fiber, hexane extraction) reveals, as disclosed in U.S. Pat. No.
5,843,499, grinding of the fiber increases both the total oil
extracted and the yield of phytosterol ferulate (mg phytosterol
ferulate/g fiber). The wt. % and ratio of campestanyl and
sitostanyl ferulates remain constant. In the case of destarching of
dry, ground corn fiber followed by extraction with hexane (Sample
4), in comparison to Sample 3, the total amount of extractable oil
increases but the yield (mg phytosterol ferulate/g fiber) and wt. %
of ferulate esters in the oil decreased. However, amylase and
protease treatment of dry, ground corn fiber followed by extraction
with hexane (Sample 2), when compared to Samples 3 and 4, increased
the total oil extracted and yield of (mg phytosterol ferulate/g
fiber) ferulate esters. However, the wt. % of ferulate esters in
the oil was still lower than Sample 3.
[0293] When the fiber was not ground and was subjected to
destarching and protease treatment followed by extraction with
hexane, Sample 1, when compared to Sample 3, the total extractable
oil, the yield of phytosterol ferulate (mg phytosterol ferulate/g
fiber), and, significantly, the wt. % of ferulate esters
(sitostanyl ferulate) increased. This data indicates that
destarching of corn fiber increases the total extractable oil while
extraction of amylase and protease treated corn fiber increases
both yields of phytosterol ferulate and total oil. Maximum benefit
is obtained by extraction of destarched and protease treated
unground corn fiber.
[0294] Because it is evident that extraction of destarched and
protease treated unground corn fiber provides significant
advantages over the methods disclosed in U.S. Pat. No. 5,843,499,
different solvents were examined to assess their effectiveness in
corn fiber oil extraction relative to a hexane solvent. As can be
seen from Table 12 above, several solvents provide increased total
extractable oil, increased yield of phytosterol ferulate (mg
phytosterol ferulate/g fiber), and, significantly, increased wt. %
of ferulate esters in the oil. The most efficient solvents tested
in this Example are ethyl acetate and acetone. These solvents
provide significant advantage over hexane in that they can be used
directly on wet corn fiber (See Examples 6 and 7.) Thus, it has
surprisingly been found that extraction of wet, unground destarched
and protease treated corn fiber, offers distinct advantage in terms
of industrial utility, enhanced yields of total extractable oil,
enhanced yield of phytosterol ferulate (mg phytosterol ferulate/g
fiber) and comparable wt. % of extracted ferulate esters in the
oil, relative to the methods disclosed in the prior art.
Example 19
Effect of Bleaching Agent Concentration on Cellulose Purity
[0295] Water wet cellulose obtained by a sequence of two caustic
extractions (2 h, 80.degree. C.; 4 h, 100.degree. C.) was added to
a 300 mL three-neck round bottom flask equipped with a reflux
condenser and mechanical stirrer. Water was added and the pH was
adjusted to 14. After heating the solution to 60.degree. C., 30%
H.sub.2O.sub.2 was added slowly. During the course of the reaction,
NaOH was added as necessary to maintain the pH at about 14. After
adding all of the H.sub.2O.sub.2, the reaction was stirred for 55
min before filtering to separate the cellulose material from the
extractant. The cellulose material was washed with four 150 mL
portions of H.sub.2O followed by washing with two 150 mL portions
of acetic acid. In Table 13 below, carbohydrate analysis of the
starting material (cellulose material after 2 alkaline extractions)
is set out in Sample 1 and the results of bleaching of this sample
using varying amounts of H.sub.2O.sub.2 under otherwise identical
conditions are set out in Samples 2 to 4.
14TABLE 13 EFFECT OF BLEACHING AGENT CONCENTRATION ON PURITY OF
CELLULOSE MATERIAL H.sub.2O.sub.2 % Yield % % % % % Sample (molar)
Cellulose Glu Xyl Gal Ara Man 1 SM SM 81.2 9.7 2.3 4.0 2.0 2 0.85
50 93.0 3.5 0.0 0.5 3.0 3 2.0 65 92.7 3.5 0.0 1.2 2.5 4 4.0 98 93.6
3.2 0.0 0.9 2.3 Notes for Table 13: SM = statring material (caustic
extracted corn fiber)
[0296] The results in Table 13 demonstrate that a wide
concentration of bleaching agent can be utilized to increase the
purity of cellulose material extracted with alkali. Surprisingly,
as the concentration of bleaching agent was increased, the recovery
of cellulose increased. Furthermore, higher concentrations of
bleaching agent were more effective at decreasing the xylose and
mannose content of the cellulose material, while all concentrations
of bleaching agent examined effectively lowered the amount of
galactose and arabinose as compared to the starting material.
Example 20
Effect of Bleaching on Derivatization of Cellulose Obtained From
Corn Fiber
[0297] The influence of bleaching agent concentration on the
ability to obtain high purity cellulose suitable for use in
derivatization reactions was examined using the following standard
procedures in which the cellulose samples correspond to those
described in Table 13 which were prepared by a sequence of two
caustic extractions (2 h, 80.degree. C.; 4 h, 100.degree. C.) and
H.sub.2O.sub.2 bleaching.
[0298] H.sub.2SO.sub.4 Procedure: Acetic acid wet cellulose was
added to a 50 mL three-neck round bottom flask equipped with a
reflux condenser and mechanical stirrer. Acetic acid, 5 wt. %
H.sub.2SO.sub.4 (based on dry weight of cellulose) and acetic
anhydride, respectively, were added to the cellulose. The reaction
was heated to 50.degree. C. and stirred for 65 minutes, which
yielded a slightly hazy solution. The resulting cellulose
triacetate was isolated by pouring the solution into water, thus
yielding a cellulose triacetate precipitate. The solids were
isolated by filtration, washed with water, and dried at 60.degree.
C. under reduced vacuum.
[0299] TFAA Procedure: Acetic acid wet cellulose was added to a 100
mL three-neck round bottom flask equipped with a reflux condenser
and mechanical stirrer. Acetic acid, acetic anhydride, and TFAA
(1.2/1/2.1 v/v/v) were added to the cellulose. The reaction was
heated to 50.degree. C. and stirred for 65 minutes, which provided
a clear solution. The resulting cellulose triacetate was isolated
by pouring the solution into water, thus yielding a cellulose
triacetate precipitate. The solids were isolated by filtration,
washed with water, and dried at 60.degree. C. under vacuum. The
results are summarized in Table 14 below. The reference to
carbohydrate composition in the Table relates to the starting
cellulose material.
15TABLE 14 COMPOSITION OF CELLULOSE DERIVATIVES (ESTERS) OBTAINED
FROM BLEACHED CELLULOSE MATERIAL % Yield % % % % % Mn Mw Mz Mw/
Sample CTA.sup.(a) Glu Xyl Gal Ara Man DS (.sup.1H) (10.sup.3)
(10.sup.4) (10.sup.5) Mn H.sub.2SO.sub.4 Procedure 1 65 93.0 3.5
0.0 0.5 3.0 2.98 8.62 3.27 0.89 3.8 2 85 92.7 3.5 0.0 1.2 2.5 2.9
9.94 4.14 1.03 4.17 3 94 93.6 3.2 0.0 0.9 2.3 3.09 8.79 3.57 0.89
4.06 TFAA Procedure 4 63 93.0 3.5 0.0 0.5 3.0 2.92 10.21 7.64 4.81
7.48 5 84 92.7 3.5 0.0 1.2 2.5 2.78 12.72 8.05 3.44 6.33 6 93 93.6
3.2 0.0 0.9 2.3 3.12 11.26 6.46 3.06 5.74 Notes for Table 14:
.sup.(a)Yields are based upon the amount of recovered triacerate
and unreacted material.
[0300] This Example reveals that the sequence of two caustic
extractions followed by bleaching provides cellulose of sufficient
purity for use in esterification reactions. The cellulose that was
bleached with 4 M H.sub.2O.sub.2 and having the highest value of
glucose (Sample 3 and 6) gave the highest yields of CTA (cellulose
triacetate). With the H.sub.2SO.sub.4 procedure, weight-average
molecular weights in the range of 33,000 to 41,000 were obtained,
while the TFAA procedure using the same cellulose gave
weight-average molecular weights in the range of 65,000 to 81,000.
In the case of H.sub.2SO.sub.4, the polydispersity was about ca.
3.8 to 4.1, while the TFAA procedure gave a much higher
polydispersity of 5.7 to 7.5. These observations indicate that TFAA
degrades the cellulose less than H.sub.2SO.sub.4 and that TFAA is
more effective in esterifying non-glucose monomers and solubilizing
any low-molecular weight polymers present.
Example 21
Effect of an Acid Step on the Purity of Cellulose Obtained From
Corn Fiber
[0301] In order to examine the effect of an acid step during the
treatment of cellulose from corn fiber, different combinations of
caustic, bleaching and acid treatments were examined. The results
are summarized in the following Table
16TABLE 15 EFFECT OF DIFFERENT COMBINATIONS OF CAUSTIC, BLEACHING
TREATMENTS AND ACID RINSES ON COMPOSITIONS OF CELLULOSE ESTERS CTA
Residue DS Mn Mw Mz Mw/ % % % % % Sample Treatment (g) (g)
(.sup.1H) (10.sup.3) (10.sup.4) (10.sup.5) Mn Glu Xyl Gal Arab Man
1 C1-C2-B1- 2.47 0 3.1 5.18 1.01 0.18 1.9 91.8 3.5 1.0 0.4 4.2
HOAc(30) 2 Cl- 2.51 0.25 3.09 7.27 2.3 0.68 3.2 93.5 2.8 0.4 0.4
2.8 H.sub.2SO.sub.4(30)- C2-B1 3 Cl- 3.82 0.43 2.98 17.9 6.54 1.95
3.6 94.7 2.0 0.0 0.0 3.3 HOAc(15)- C2-B1 4 Cl- 2.47 0.21 3.04 38.31
7.9 1.36 2.06 95.2 2.1 0.0 0.0 2.7 HOAc(15)- B1 5 Cl- 2.47 0.05
3.07 5.32 2.15 0.73 4.05 95.4 2.2 0.0 0.0 2.4 HOAc(15)- B2 Notes
for Table 15: Cl = Caustic treatment of destarched corn fiber at
80.degree. C., 2 h. C2 = Second caustic treatment of cellulose
fiber obtained from corn fiber at 80.degree. C. 2 h. HOAc(30) =
Treatment with refluxing acetic acid for 30 min. HOAc(15) =
Treatment with refluxing acetic acid for 15 min. B1 = Bleaching at
60.degree. C., 1 h. B2 = Bleaching at 80.degree. C., 2 h.
Acetylation with 5% H.sub.2SO.sub.4, Ac.sub.2O, HOAc.
[0302] The results above illustrate that inclusion of an acid step
can significantly decrease the amount of unreacted starting
material, which greatly improves the clarity of the reaction
mixture and facilitates filtration of the reaction mixture prior to
precipitation. Furthermore, the molecular weights of the CTA were
high, ranging from about 10,000 to 80,000. When the acid step
followed a caustic step and preceded the bleaching step (compare
Samples 1, 4 and 5), inclusion of an acid step significantly
increased the glucose content (purity) measured in the CTA. The
values of 95% glucose for Samples 4 and 5 are among the highest
observed for cellulose esters made from corn fiber cellulose.
Example 22
Effect of Xylanase Treatment on Purity of Cellulose
[0303] In order to explore the possibility of further purifying
cellulose from corn fiber by enzymatic pulping e.g., treatment with
a xylanase enzyme, the following experiment was performed.
[0304] A 100-mL round-bottomed flask equipped with an overhead
mechanical stirrer, was charged with 5 g of cellulose material
produced from a single caustic extraction (80.degree. C., 2.5 hours
of corn fiber and 40 mL of water. The pH of the mixture at this
point as determined by pH paper was around pH 6. The mixture was
heated to 60.degree. C. and 5.0 mL of xylanase was added to the
mixture. The mixture was stirred at 60.degree. C. for 15 minutes
and an additional 20 mL of water was added. The mixture was stirred
at 60.degree. C. for 90 additional minutes. The cellulose material
was separated by filtration and was washed with about 200 mL of
water.
[0305] Sample 1 was split into two portions. One portion of the
solid was dried in a vacuum oven at 50.degree. C. overnight to
produce Sample 2 in Table 16 and was submitted for X-ray, GPC, and
carbohydrate analyses. A 100-mL round-bottomed flask equipped with
an overhead mechanical stirrer was charged with the remaining
portion of the moist cellulose material and 50 mL of water. The
mixture was heated to 70.degree. C. with stirring for 30 minutes
before adding 2.0 g of NaOH to the mixture. The reaction was then
heated to 80.degree. C. and held at this temperature with stirring
for 120 minutes. The cellulose material was isolated by filtration
at or above 60.degree. C. The filtercake was washed with about 500
mL of water at 60.degree. C., 200 mL of 20 vol. % HOAc and an
additional 300 mL of water. The cellulose material was dried at
50.degree. C. in a vacuum oven to produce Sample 3 and was
submitted for X-ray, GPC, and carbohydrate analyses.
17TABLE 16 EFFECT OF XYLANASE TREATMENT ON CELLULOSE PURITY Treat-
% Yield Total % % % % % Xyl/ Mw/ Sample ment.sup.1 Cellulose.sup.2
Carb Glu Xyl Gal Ara Man Ara M.sub.w.sup.3 M.sub.n.sup.3 Mn.sup.3 1
Cl 17.8 89 75.3 13 2.6 6.8 2.7 2 nd Nd nd 2 Cl-X 12.8 89 91.1 4.2
1.1 1 2.7 4 171,496 90,209 1.9 3 Cl-X- 10.7 100 93.1 3 0.5 0.7 2.7
4 152,610 31,262 4.9 C2 Notes for Table 16: .sup.1Cl = NaOH, for
2.5 h at 80.degree. C.; Cl-X = NaOH, for 2.5 h at 80.degree. C.
then Multifect Xylanase for 1.75 h at 60.degree. C.; Cl-X-C2 =
NaOH, for 2.5 h at 80.degree. C. then Multifect Xylanase for 1.75 h
at 60.degree. C. then NaOH for 2 h at 80.degree. C. .sup.2Total
yield of cellulose from 250 g of dried corn fiber. .sup.3GPC
results for cellulose as determined from cellulose
tricarbailate.
[0306] These results show that the purity of the corn fiber
cellulose can be significantly improved by treating the cellulose,
obtained by a single caustic extraction, with a xylanase enzyme. In
this case, the glucose content (purity) increased from 75% to 91%.
A second caustic extraction of the xylanase treated cellulose
increased the purity slightly to 93% glucose. As the samples
illustrate, the molecular weight of the cellulose was high which,
when combined with the purity, makes this cellulose suitable for
derivatization.
Example 23
Effect of Bleaching Treatment on Arabinoxylan and Cellulose
[0307] A series of bleachings experiments were performed to examine
the effect of bleaching at various times in the alkaline extraction
process.
[0308] 10 g of destarched corn fiber (about 3.0 mmol of
polysaccharide based upon the typical yield of cellulose and
arabinoxylan) were added to a solution of NaOH at 80.degree. C. in
a 500 mL three-neck glass round bottom flask equipped with a
mechanical stirrer and reflux condenser. The reaction was stirred
for 30 min at 80.degree. C. In Sample 1, no bleaching agent was
added. In Sample 2, about 2.4 molar equivalents of H.sub.2O.sub.2
were added to the destarched corn fiber according to the method of
Doner et al. disclosed in WO 98/40413. Very vigorous bubbling and
frothing occurred during this reaction, thus requiring the exercise
of extreme caution during the process. In Sample 3, after
extraction with alkali, the cellulose material was allowed to cool
to room temperature before adding 2.4 molar equivalents of
H.sub.2O.sub.2 and allowing the cellulose material to bleach for 30
min. Sample 4 was processed similarly to Sample 3 except that
bleaching was for 95 min. For comparison purposes, arabinoxylan,
isolated directly by precipitation in acetic acid, from the first
caustic extraction of arabinoxylan (2 h, 80.degree. C.) and the
corresponding bleached cellulose (Example 19) are included in Table
17 as Sample 5.
[0309] In each of the samples, the alkali extracted corn fiber was
filtered to remove the alkali extractant containing arabinoxylan
and the remaining cellulose material was washed with water. The
alkali extractant was collected. The cellulose material was dried
at 60.degree. C. under house vacuum. The extracted filtrate was
combined from the rinsings and the pH of the combined filtrate was
adjusted to 5 followed by filtering through celite. Ethanol (about
3/1 ROH/filtrate liquids) was added to the filtrate, resulting in a
gummy precipitate that was separated by filtration. When nearly all
liquid had been removed from the gummy precipitate, fresh alcohol
was added to harden the solid to provide a precipitate containing
arabinoxylan. The arabinoxylan samples were then dried at
60.degree. C. under reduced pressure.
18TABLE 17 EXAMINATION OF ARABINOXYLAN AND CELLULOSE COMPOSITION AS
A FUNCTION OF BLEACHING AND PRECIPITATION AGENT % Glu % Xyl % Gal %
Ara % Man % Arabino- Arabino- Arabino- Arabino- Arabino- Glu % Xyl
% Gal % Ara % Man Entry xylan xylan xylan xylan xylan Cell Cell
Cell Cell Cell 1.sup.(a) 8.1 51.1 9.1 31.7 0.0 81.4 8.5 2.0 4.7 3.5
2.sup.(a) 11.0 52.5 6.1 30.4 0.0 89.1 4.6 1.0 1.7 3.6 3.sup.(a) 7.5
50.7 8.8 32.9 0.0 82.1 8.3 2.0 4.4 3.1 4.sup.(a) 8.0 48.4 8.7 34.6
0.2 78.7 10.8 2.2 5.0 3.2 5.sup.(b) 2.9 53.2 8.2 35.7 0.0 93.6 3.2
0.0 0.9 2.3 Notes for Table 17 Isolated by precipitation with
alcohol. .sup.(b) Isolated by precipitation with acetic acid. Ara =
Arabinose. Cell = cellulose.
[0310] The above table illustrates that the composition of
arabinoxylan obtained by the method disclosed by Doner et al.
(Sample 2) is different from that of the arabinoxylan obtained by
bleaching after alkaline extraction or by omitting bleaching of the
arabinoxylan altogether. In particular the arabinoxylan of Sample 2
has a higher level of glucose and lower level of arabinose.
Bleaching after alkaline extraction but before isolation of the
arabinoxylan had only a marginal influence on the arabinoxylan, as
the composition of the arabinoxylan in Samples 3 and 4 are similar
to Sample 1 where the reaction conditions and isolation procedure
was the same and there was no bleaching step. The differences in
composition of the arabinoxylan are particularly pronounced when
Sample 2 is compared to Sample 5 (alkaline extraction, no
bleaching, isolated by acetic acid precipitation) which is
illustrative of a preferred practice of this invention.
[0311] When the cellulose material obtained from the caustic
extraction was examined, the process of Doner et al. (Sample 2) led
to a higher percentage of glucose in the cellulose fraction
relative to Samples 1, 3, and 4. Bleaching after extraction but
before separation (compare Samples 1, 3 and 4) had no significant
influence of the purity (wt. % glucose) of the cellulose. Bleaching
of the cellulose after separation of the alkali extractant (Sample
5) significantly improved the purity of the cellulose.
[0312] To further illustrate the impact of bleaching upon the
arabinoxylan structure, the composition and molecular weight of the
arabinoxylan prepared according to the method of Donner et al. was
compared to those obtained under different conditions (no
bleaching) which are summarized below.
19TABLE 18 COMPARISON OF ARABINOXYLAN COMPOSITION AS A FUNCTION OF
ALKALI/BLEACHING TREATMENT CONDITIONS Base Time Temp % % % % %
Xyl/Ara Mn Mw Mz Mw/ Sample M (h) (.degree. C.) Glu Xyl Gal Ara Man
Ac (10.sup.3) (10.sup.4) (10.sup.5) Mn 1 2.5 0.5 80 11.0 52.5 6.1
30.4 0.0 1.7 60.98 27.65 12.77 4.53 NaOH 2 2.5 0.5 80 7.1 50.2 9.2
33.3 0.1 1.5 105.1 40.49 13.6 3.85 NaOH 3 2.5 0.5 80 6.5 49.9 7.4
33.3 2.9 1.5 119.6 41.2 20.5 3.45 NaOH 4 2.5 1 80 7.9 50.9 9.1 32.1
0.0 1.6 92.65 34.81 9.79 3.76 NaOH 5 2.5 2 80 7.1 51.5 9.0 32.5 0.0
1.6 105.5 38.85 11.95 3.68 NaOH 6 2.5 2 80 2.9 53.2 8.2 35.7 0.0
1.5 121.2 41.67 21.01 3.44 NaOH
[0313] The arabinoxylan of Sample 1, prepared according to the
method of Doner et al. as discussed above, has a weight-average
molecular weight of 276,500. In the case of the arabinoxylan that
was not subjected a bleaching step (Samples 2-6), the
weight-average molecular weights were significantly higher (416,700
to 348,100) and the samples exhibited lower polydispersity as
compared to Sample 1. This surprising result is critical to the
invention because it is highly desirable to have high molecular
weight polymers with low polydispersity for most applications.
These results demonstrate that bleaching of the cellulose fiber
prior to separation of the arabinoxylan component, as performed
according to the method of Doner et al., has undesirable effects on
the molecular weight of the arabinoxylan obtained.
[0314] To further explore the effects of bleaching on the color of
aqueous solutions of arabinoxylan, the yellowness index color of
arabinoxylan samples were compared. In the case of Sample 1,
precipitation with ethanol gave a brown colored solid. The
arabinoxylan from Samples 2 to 4 were semi-white. Further,
precipitation of the arabinoxylan of Sample 1 in acetic acid, as
set forth in Sample 5, provided a semi-white solid comparable in
color to Samples 2 to 4 after alcohol precipitation. Further
precipitation of Samples 2 to 4 in acetic acid improved the color
relative to that obtained by alcohol precipitation alone. Relative
physical property data is summarized in Table 19 below.
20TABLE 19 YELLOWNESS INDEX OF ARABINOXYLAN SOLUTIONS Reaction Temp
YI Color YI Color Sample Base (M) Time (h) (.degree. C.) (ROH ppt)
(HOAc ppt) 1 2.5 0.5 80 1449 1096 NaOH 2 2.5 0.5 80 831 463 NaOH 3
2.5 0.5 80 692 466 NaOH 4 2.5 0.5 80 611 410 NaOH 5 2.5 2 80 nd nd
NaOH
[0315] Sample 2 is the arabinoxylan obtained according to the
method of Doner et al. When precipitated in alcohol, this material
had a YI (yellowness index) of 831. Subsequent precipitation of the
arabinoxylan in Sample 2 with acetic acid improved the color of
that sample (YI of 831 vs. 463 after acetic acid treatment). The
samples bleached at RT (room temperature) after alkaline
extraction, but before separation of the components, were less
yellow than Sample 2 (compare Samples 3 and 4 with Sample 2).
[0316] This data illustrates that the method of Doner et al. yields
an arabinoxylan with a different composition (higher glucose, lower
arabinose), lower molecular weight, and an unacceptable (non-white)
color. The method of Doner et al. does increase the purity of the
cellulose somewhat; however, the purity provided by the Doner et
al. method does not result in a reaction grade cellulose.
[0317] Significantly, the method of Doner et al. is not safe to
practice on an industrial scale. In contrast, the methods of the
present invention do not create unmanageable dangerous conditions.
Thus, the methods of this invention are practicable on an
industrial scale.
Example 24: Esterification of Cellulose Obtained From Corn
Fiber
[0318] Three hundred grams of non-dried corn fiber were placed in a
porous bag in a soxhlet extractor and extracted with ethanol for
about 15 hours. The ethanol was removed and the corn fiber was
extracted with diethyl ether for about 7 hours. The corn fiber was
dried in a forced air oven at 60.degree. C. providing 277.7 g of
corn fiber. Concentration of the ethanol fraction gave 6.7 g of
material and the ether fraction provided 2.7 g.
[0319] A 5000 mL three-neck glass round bottom flask equipped with
a mechanical stirrer and reflux condenser was charged with 275 g of
the extracted corn fiber, 1986 mL H.sub.2O, and 220 mL of a 1 M
phosphate buffer solution. The mixture was heated to 80.degree. C.
before adding 11 mL of Spezyme suspension. No starch could be
detected after 55 minutes. The fiber was isolated by filtration,
washed with water, and dried to obtain 191.2 g of destarched corn
fiber (69.5% yield).
[0320] 1514 mL of water was added to a 5 L three-neck round bottom
flask equipped with a reflux condenser and mechanical stirrer. The
water was heated to 80.degree. C., and 189.2 g of the destarched
corn fiber obtained above and 151 g of NaOH was added,
respectively. The reaction was stirred for 2 h at 80.degree. C.
before filtering at 65.degree. C. to remove the cellulose. The
cellulose was washed with two 200 mL portions of water preheated to
65.degree. C. The cellulose was transferred to the 5 L vessel
containing 1514 mL of water preheated to 90.degree. C. Addition of
NaOH was not necessary because the solution maintained a pH of 14.
The reaction was heated to 100.degree. C. and stirred for 4 hours.
The reaction was filtered at 65.degree. C. to remove the cellulose.
The cellulose was washed with two 200 mL portions of water
preheated to 65.degree. C. The cellulose was again transferred to
the 5 L reaction vessel containing 491 mL of water preheated to
60.degree. C. To this mixture was slowly added 66 mL of 30%
H.sub.2O.sub.2 (2 equivalents). The pH of the solution was adjusted
to 14 by the addition of 40 g NaOH. The reaction was stirred for 1
hour at 60.degree. C. then filtered at 65.degree. C. to remove the
cellulose. The cellulose was washed with two 200 mL portions of
water preheated to 65.degree. C. followed by additional acetic acid
to adjust the pH and to remove the water. A portion of the
cellulose was removed and dried to determine the % solids and the
yield was found to be 31.1 g (16.4% cellulose from the destarched
corn fiber). X-ray analysis of cellulose samples after each stage
of reaction revealed that the cellulose was maintained as cellulose
I.
[0321] The cellulose obtained above was reacted with acetic
anhydride according to the general methods described above in
Example 20.
21TABLE 20 EXAMINATION OF CELLULOSE ESTERS PREPARED USING DIFFERENT
CATALYSTS % Yield Reaction DS Mn Mw Mz Mw/ Sample CTA.sup.(a) Time
(min) (.sup.1H) (10.sup.3) (10.sup.4) (10.sup.5) Mn H.sub.2SO.sub.4
Procedure 1 65 145 2.8 12.15 6.16 1.64 5.07 TFAA Procedure 2 48 70
2.84 15.72 15.26 8.1 9.7 Notes for Table 20: .sup.(a)Yields are
based upon the amount of recovered triacetate and unreacted
material.
[0322] The yield of CTA is that expected for bleaching of the corn
fiber cellulose with 2 molar equivalents of bleaching agent (cf.
Example 20). As these entries illustrate, weight-average molecular
weights in the range of 62,000 to 153,000 can be expected from the
cellulose samples depending upon the reaction conditions.
[0323] This Example, which encompasses 2 caustic extractions and a
bleaching step, illustrates that if the cellulose fiber at each
stage of reaction is handled hot and not allowed to cool below
about 60.degree. C. during filtration while at elevated pH, the
morphology of the cellulose can be maintained as cellulose I. This
point is particularly apparent when compared to the cellulose of
Example 8 where the cellulose samples were allowed to cool to room
temperature prior to filtration.
Example 25
Concentration of Arabinoxylan Solution and Precipitation
Parameters
[0324] Portions of the filtrate from the first caustic extraction
of Example 24 were concentrated by heating the sample under vacuum
while providing for an airflow to the surface of the solution being
concentrated. By this method, foaming of the solution during
concentration is minimized. Otherwise, it was found necessary to
acidify the solution with an acid such as H.sub.2SO.sub.4 or HCl,
which leads to the formation of inorganic salts, which remain in
the arabinoxylan during precipitation and washing. As an example,
about 927 g of the filtrate from the first caustic extraction was
concentrated to provide 596 g of distilled water and 330 g of a
viscous liquid containing arabinoxylan (about 70 wt. % of the total
reaction liquids and wash were removed). The viscous liquid (heated
to 50.degree. C. to 60.degree. C.) was added slowly to rapidly
stirred cold acetic acid. According to this method, 71.9 g of
arabinoxylan was isolated (38 wt. % yield from destarched corn
fiber). Alternatively, acetic acid can be added directly to the
caustic solution as required to reduce foaming, allowing for more
rapid concentration. The sodium acetate that forms does not remain
in the arabinoxylan to any great extent (0.1-5 wt. %) as long as
acetic acid is utilized to precipitate the arabinoxylan.
[0325] This Example also illustrates that by the method described
above, it is possible to significantly concentrate the
arabinoxylan-containing solution obtained from caustic extraction
of corn fiber without having to acidify with an inorganic acid
prior to adding the solution to acetic acid. This serves to both
lower the pH and acts as a non-solvent for the arabinoxylan. By use
of this method, arabinoxylan that is not contaminated with
inorganic salts is obtained and the volume of acetic acid required
to precipitate the arabinoxylan is significantly reduced.
Example 26
Concentration of the Arabinoxylan Solution With Recovery of Caustic
by Ultrafiltration
[0326] A solution of arabinoxylan obtained by caustic extraction of
destarched and proteolyzed corn fiber was passed through a membrane
having a molecular weight cutoff of 20 kD. The membrane unit was a
M204-SW Membrane Pilot System with a 10 gal feed tank (LCI
Corporation, Charlotte, N.C.). The membrane (CONSEP) was obtained
from North Carolina SRT, Inc. and the geometry of the membrane was
a flat sheet. The number of test cells was two which were arranged
in parallel providing for 162 cm.sup.2 of cell area. The flow rate
was 1.1 grams per minute and the system pressure was 100 psi. The
total processing time was 12.3 h. The results are summarized in
Table 21 in which retentate is the fraction of the feed which did
not pass through the membrane and permeate is the fraction of the
feed which passed through the membrane.
22TABLE 21 CONCENTRATION OF ARABINOXYLAN AND NAOH IN THE RETENTATE
AND PERMEATE AFTER ULTRAFILTRATION. Arabinoxylan NaOH Total Total
Weight weight Mass % of weight Mass % of (g).sup.1 % (g) Feed % (g)
Feed Feed 3111.75 1.3 40.5 -- 3.38 105.2 -- Retentate 359.91 4.5
16.2 40.0 2.83 10.2 9.7 Permeate 2369.19 <0.1 <2.4 <5.9
2.83 67.0 63.7 Notes for Table 21: .sup.1Due to sampling during the
experiment and the material retained in the system, the total
weight of the retentate and permeate does not equal the weight of
the initial feed.
[0327] This Example demonstrates that when a 1.3 wt. % arabinoxylan
caustic solution is passed through the membrane, the solution is
concentrated to 4.5 wt. % arabinoxylan in the retentate
representing 40% of the mass of the arabinoxylan in the original
feed solution. The wt. % of NaOH is reduced from 3.38 wt. % in the
feed solution to 2.83 wt. % in the retentate. The permeate contains
63.7 wt. % of the NaOH in the original feed solution. Thus, this
Example illustrates that the caustic passes freely through the
membrane while the arabinoxylan is concentrated in the retentate
thus demonstrating that ultrafiltration is an efficient means of
recovering caustic and concentrating the arabinoxylan.
Example 27
The Effect of Velocity Across the Membrane on Concentration of the
Arabinoxylan Solution With Recovery of Caustic by
Ultrafiltration
[0328] A solution of arabinoxylan obtained by caustic extraction of
destarched and proteolyzed corn fiber was passed through a membrane
having a molecular weight cutoff of 100 kD. The membrane unit was a
M204-SW Membrane Pilot System with a 10 gal feed tank (LCI
Corporation, Charlotte, N.C.). The membrane (CONSEP) was obtained
from North Carolina SRT, Inc. and the geometry of the membrane was
a flat sheet. The number of test cells (162 cm.sup.2 of cell area)
was two, each of which was arranged in series which served to
increase the velocity. The flow rate was 1.1 grams per minute and
the system pressure was 150 psi. The total processing time was 4.7
h. Following completion of the filtration, a 5% caustic solution
was circulated across the surface of the membrane for 20 minutes to
remove any arabinoxylan deposited on the membrane. The results are
summarized in Table 22 in which retentate is the fraction of the
feed which did not pass through the membrane and permeate is the
fraction of the feed which passed through the membrane.
23TABLE 22 CONCENTRATION OF ARABINOXYLAN AND NAOH IN THE RETENTATE
AND PERMEATE AFTER ULTRAFILTRATION. Arabinoxylan Weigbt (g).sup.1
weight % Total Mass (g) % of Feed Feed 2003.5 1.3 26.0 -- Retentate
394.2 5.1 20.1 77.3 Caustic Wash 104.25 3.9 4.1 15.8 Permeate
1609.3 <0.1 <1.6 <6.2 Notes for Table 22: .sup.1Retentate
mass was determined by difference (feed mass less permeate
mass)
[0329] This Example demonstrates that when a 1.3 wt. % arabinoxylan
caustic solution is passed through the membrane, the solution is
concentrated to 5.1 wt. % arabinoxylan in the retentate
representing 77.3% of the mass of the arabinoxylan in the original
feed solution. An additional 15.8% of the arabinoxylan was
recovered in the caustic wash. Thus, this Example illustrates that
the caustic passes freely through the membrane while the
arabinoxylan is concentrated in the retentate. At the higher
velocity and concentration, some arabinoxylan is deposited on the
membrane but is easily removed by washing the surface of the
membrane.
Example 28
Esterification of Arabinoxylan
[0330] 4 g (dry weight basis) of arabinoxylan obtained by caustic
extraction of destarched corn fiber (two conditions, 100.degree.
C.; 4 hours or 0.5 hours, 80.degree. C.) was added to a 100 mL
three-neck round bottom flask equipped with a reflux condenser and
mechanical stirrer. The arabinoxylan was isolated by one of two
methods: (1) ethanol precipitation followed by drying (this
arabinoxylan was used with no special pretreatment); or (2) by
acetic acid precipitation, (this arabinoxylan was added to the
reaction vessel wet with acetic acid). The appropriate amounts of
acetic anhydride, acetic acid, and catalyst (H.sub.2SO.sub.4 or
TFAA) were added to the arabinoxylan. The reaction was then stirred
at 50.degree. C. for the indicated times. The reaction was filtered
to remove any unreacted solids. The unreacted solids were dried to
obtain their weights. The filtrate was added to water and any
resulting solids were washed with three 25 mL portions of water and
dried. The results are summarized in the following Table.
24TABLE 23 CHARACTERISTICS OF ESTERIFIED ARABINOXYLAN BASED UPON
PRECIPITATION CONDITIONS AND REACTION CONDITIONS Reaction Acylated
Recovered Con- arabino- arabino- DS Mn Mw Mz Mw/ Tg T.sub.decomp
Sample ditions xylan (g) xylan (g) (.sup.1H) (10.sup.3) (10.sup.4)
(10.sup.5) Mn (.degree. C.) (.degree. C.) 1 TFAA/Ac.sub.2 2.66 1.03
2.73 4.84 2.41 1.56 4.99 79 225 O/HOAc, 24.0 h (a,c) 2
TFAA/Ac.sub.2 0.88 2.37 2.86 6.56 4.29 2.81 6.55 101 220 O/HOAc,
27.7 h (a,d) 3 5% H.sub.2SO.sub.4/Ac.sub.2 0 3.62 0 0 0 0 0 0 0
O/HOAc, 22.8 h (a,c) 4 5% H.sub.2SO.sub.4Ac.sub.2 0.68 0 2.07 1.9
0.38 0.12 1.97 71 220 O/HOAc, 1.8 h (b,c) 5 TFAA/Ac.sub.2 3.97 0
2.35 26.88 11.05 4.1 4.11 99 225 O/HOAc, 1.0 h (b,c) Notes for
Table 23: (a) The arabinoxylan was precipitated using ethanol,
dried and used without further treatment. (b) The arabinoxylan was
precipitated from HOAc and used directly without drying. (c) 4 h,
100.degree. C. extraction. (d) 0.5 h, 80 .degree. C.
extraction.
[0331] This Example reveals that TFAA can effectively catalyze the
esterification of arabinoxylan without significant molecular weight
loss compared to a catalyst such as sulfuric acid (compare Sample 4
with Sample 5). Furthermore, this Example reveals that arabinoxylan
esters are more easily prepared when the arabinoxylan is used
directly after precipitation in acetic acid and not dried. That is,
in a preferred practice of this invention, the arabinoxylan will be
precipitated in acetic acid to obtain better color without the
difficulties of inorganic salt generation. With the methods of this
invention, there is a surprising additional advantage that the
arabinoxylan is obtained in a more reactive form.
Example 29
Etherification of Arabinoxylan
[0332] H.sub.2O (15 mL) was added to an arabinoxylan (2 g) obtained
by caustic extraction (1.5 M, 80.degree. C., 2 h) of destarched
corn fiber. After stirring for 15 minutes, KOH (1.6 g) was added,
and the solution stirred an additional 15 minutes. Methyl iodide (9
mL) was added and the reaction was stirred 43 hours at room
temperature. The reaction pH was adjusted to 6 with HCl before
diluting the solution with H.sub.2O (10 mL). The solution was
warmed to about 50.degree. C. before adding ethanol (225 mL)
slowly. Upon addition of the ethanol, all of the salts dissolved.
Completion of the ethanol addition allowed precipitation of the
arabinoxylan methyl ether. The solid was filtered, shaken with warm
ethanol (220 mL, about 50.degree. C.) filtered and washed with EtOH
(150 mL). The product was dried at 55.degree. C. at reduced
pressure providing 1.95 g of arabinoxylan methyl ether. Carbon 13
NMR and .sup.1H NMR (FIG. 8) were consistent with formation of the
expected arabinoxylan methyl ether. Thermal analysis revealed that
the ether was amorphous, having a Tg of 161.degree. C. and that the
ether was thermally stable to 275.degree. C.
Example 30
Effect of Arabinoxylan Precipitation Parameters on
Derivatization
[0333] Dried crude arabinoxylan obtained from the caustic
extraction of corn fiber was activated with H.sub.2O (27 mL
H.sub.2O per 1 g of arabinoxylan). The aqueous solution was then
exposed to AcOH (37 mL per 1 g of arabinoxylan) in order to
precipitate arabinoxylan. After filtering the arabinoxylan, it was
washed with AcOH (3.times.10 mL per 1 g of arabinoxylan) (Samples 1
to 6) or an equivalent amount of propionic acid (Sample 7). The wet
arabinoxylan was then exposed to an appropriate amount of Ac.sub.2O
and TFAA (Samples 1 to 6 and 8) or propionic anhydride and TFAA
(Sample 7). Alternatively, the wet arabinoxylan was exposed to an
appropriate amount of Ac.sub.2O and 0.7% H.sub.2SO.sub.4 (Sample
9). The reaction was stirred for 1 hour (after addition of the
reagents) and the product was isolated by precipitation from water
unless otherwise stated. The white solids were washed with H.sub.2O
until the filtrate was neutral pH and the product was dried in
vacuo. The results are summarized in Table 24.
25TABLE 24 EFFECT OF ARABINOXYLAN SALT CONTENT ON DERIVATIZATION
Recovered Esterified Reaction DS Mn Mw Mz Mw/ Tg T.sub.decomp
T.sub.90% decomp Sample Substrate Substrate Product Temp (.degree.
C.) (.sup.1H) (10.sup.3) (10.sup.4) (10.sup.5) Mn (.degree. C.)
(.degree. C.) (.degree. C.) 1 A, 4.0 g 3.3 g 0.3 g 50 -- -- -- --
-- -- -- -- 2 B, 9.1 g, 0 4.8 g, 0.3% 49 2.36 18.5 28.1 10 15.2 130
250 319 56.6% salt salt 3 A + B, 4.0 g, 0 2.2 g, 54 2.46 31.2 17.3
5.48 5.54 124 220 284 57.8% salt 11.7% salt 4 A + B, 4.0 g, 0 2.2
g, 64 2.5 15.2 6.76 2.56 4.46 129 200 279 57.8% salt 15.8% salt 5 A
+ B, 4.0 g, 0 3.5 g, 55 2.4 23.2 21.0 6.68 9.04 120 200 285 38.8%
salt 14.0% salt 6 A + B, 60.0 g, 0 52.6 g 50 2.5 32.6 17.8 5.85
5.48 125 200 283 38.8% salt 14.8% salt 7 A + B, 4.0 g, 0 3.7 g, 55
2.36 22.4 16.7 6.19 7.45 94 200 298 38.8% salt 12.4% salt 8 B, 4.0
g, 0 5.7 g, 50 2.36 27.4 48.6 21.3 17.7 125 240 308 0.1% salt 0.1%
salt 9 B, 4.0 g, 0 3.3 g 50 2.51 10 6.09 2.69 6.09 0.1% salt Notes
for Table 24: A = arabinoxylan A; B = arabinoxylan B; A + B = both
arabinoxylan (hemicellulose) A and B in their naturally occurring
proportions; entry 2 was precipitated from 10:90 AcOH/H.sub.2O;
entry 7 was reprecipitated from an acetone solution into 20/80
methanol/H.sub.2O; % salt indicates the weight % of the sample
which was not readily combustible.
[0334] This Example reveals a number of important aspects
concerning arabinoxylan isolation and esterification. As previously
described, a preferred method of isolation of arabinoxylan from
corn fiber is via AcOH precipitation because this method results in
a white product and a more satisfactory carbohydrate balance than
does the sample obtained via alcohol precipitation.
[0335] AcOH precipitation can be carried out in two ways. In the
one method, the aqueous arabinoxylan solution is added to cold AcOH
quickly resulting in the rapid precipitation of a polysaccharide
ultimately having a high salt content (as high as 60%). This high
salt content arabinoxylan was used in Samples 1 to 7. However in a
more preferred method of precipitation, room temperature AcOH is
added to the aqueous solution of arabinoxylan allowing for a slower
precipitation of the polysaccharide resulting in greatly diminished
salt contents (0.1 to 2%). Low salt content arabinoxylan was
utilized in Samples 8 to 9.
[0336] Significantly, when the pH of the solution is lowered slowly
during precipitation it is possible to separate two components. The
first and minor component (hemicellulose A) precipitates from
solution at about pH 4 to 4.5. This material has a normalized
glucose content of approximately 38%. More notably, once
hemicellulose A is dried it is no longer soluble in water at acidic
or neutral pH. The pH must be adjusted to at least 12 to 13 before
the polysaccharide will go into solution. The second and major
component that precipitates (hemicellulose B) contains almost no
glucose (1% or less) and, in contrast to hemicellulose A, is fully
water soluble in aqueous solution ranging from mildly acidic to
strongly alkaline (even after drying). The nature of the
hemicellulose plays a role in its ability to be esterified.
[0337] Sample 1 shows that once dried, the hemicellulose A of
arabinoxylan is not readily esterified. This may be due in part to
insufficient activation. Even though the hemicellulose A fibers
swell when they come into contact with water they do not completely
dissolve. Sample 2 shows that the hemicellulose B component is
readily esterified.
[0338] Furthermore, it is not necessary to have purified
arabinoxylan prior to esterification to produce "salt free"
product. Starting with material that was almost 60% salt by weight,
product was isolated having only 0.3% salt content. In the case of
the mixed hemicellulose esters (A+B), reprecipitation has little
influence on salt content. In general, esterification of mixed
hemicelluloses results in products with higher salt content than
when the esterifications are carried out on hemicellulose B only.
This may be due to insufficient activation of the A component which
leads to salts being trapped within the A component. Of additional
interest in Sample 2 is the fact that with low salt content, the
polymer shows much improved thermal stability with an initial
decomposition temperature of 250.degree. C., as compared to the
much lower values for the higher salt content polymers. This
property has a significant impact on applications such as thermal
casting and extrusion. Sample 4 shows that as the temperature of
the acylation reaction is increased over 55.degree. C. significant
molecular weight breakdown occurs. Sample 7 shows that this
acylation methodology can be readily extended to other esters such
as arabinoxylan propionates.
[0339] The best results, as judged by acetyl weight gain, are
apparent when utilizing arabinoxylan of greater purity i.e., low
salt content, as shown in Sample 8. Sample 9 shows that lower salt
content in the arabinoxylan also allows for the use of the
H.sub.2SO.sub.4 catalyzed method of esterification. Sample 9 also
illustrates the ability to isolate arabinose and xylan via
acetolysis of the arabinoxylan. After acetylation under these
conditions, the polysaccharide ester was found to contain 72.3%
xylose and only 7.5% arabinose with the arabinose, being released
as arabinose acetate.
Example 31
Hydrolysis of Arabinoxylan
[0340] A solution of 2 g of corn fiber arabinoxylan (obtained by a
single caustic extraction with 2 M NaOH at 100.degree. C. and
isolated by precipitation in acetic acid) in 10 mL of 80/20
water/acetic acid containing 100 mg of sulfuric acid was heated to
100.degree. C. Initially, the solution was highly viscous but the
viscosity dropped very dramatically after about 35 min. Samples
were removed at 2 hours 15 min, 3 hours 45 min, and 6 hours 45 min.
The pH of each solution was adjusted to 5-6 using 4 M NaOH and the
solutions were concentrated to dryness. Carbon 13 NMR spectra of
each sample revealed that hydrolysis was essentially complete after
6 hours 45 min. The sample corresponding to hydrolysis for 6 hours
45 min (950 mg) in 2 mL of water was filtered to remove solids and
the filtrate was concentrated. Analysis by .sup.1H NMR showed that
the removed solid (20 mg) corresponded to oligomeric starting
material while the carbohydrates comprising the filtrate (890 mg)
consisted essentially only of xylose and arabinose (the 500 MHz
.sup.1H spectra is shown in FIG. 9). To remove salts, the filtrate
was taken up in 3 mL of water and passed through a mixed ion
exchange column (6.5 cm.times.1.0 cm, Bio-Rad AG501-X8) which was
washed with four 3 mL portion of water. After concentration of the
combined liquids, 670 mg of arabinose and xylose was obtained.
[0341] This Example reveals a number of important features of this
invention. First, the corn fiber arabinoxylan isolated by
precipitation in acetic acid can be carried directly to the
hydrolysis step without having to remove the acetic acid from the
arabinoxylan. This is extremely beneficial in an industrial
process. Further, because corn fiber arabinoxylan has a higher
solubility than arabinoxylan obtained from other sources, such as
wood arabinoxylan, higher solids can be obtained in the hydrolysis
step, which is also industrially useful. Furthermore, any unreacted
solids can be removed by a simple filtration, which greatly aids in
the purification process.
Example 32
Selective Hydrolysis of Arabinoxylan
[0342] A solution of 5 g of corn fiber arabinoxylan, obtained by a
single caustic extraction with 2 M NaOH at 100.degree. C. and
isolated by precipitation in acetic acid, in 25 mL of D.sub.2O
containing 250 mg of sulfuric acid was heated to 58.degree. C.
Initially, the solution was highly viscous but the solution
viscosity dropped very quickly. Samples were removed at 1 hour 30
min, 3 hours 15 min, 5 hours 30 min, 7 hours 50 min, and 23 hours
30 min. Each solution was neutralized with 3 M NaOH and carbon 13
NMR spectra of each sample were collected, which are shown in FIG.
10 along with that of the starting material. These spectra
demonstrate that arabinose is hydrolyzed much faster than
xylose.
[0343] 4.0 mL of ethanol was added to about 4.5 mL of the sample
removed at 5 hours 30 min, providing a gummy solid. The solid was
isolated by filtration, washed with ethanol and dried to give 614
mg of filtrate. The filtrate was concentrated, providing 150 mg of
an oil. Proton NMR of the oil revealed that the oil consisted of
arabinose contaminated with a small amount of xylose. Pure
arabinose was isolated from the oil by crystallization from 90/10
ethanol/water (FIG. 11). Carbon 13 NMR of the solids isolated by
ethanol precipitation revealed that they consisted essentially of
xylan, with little arabinose present.
[0344] This Example reveals that, due to the higher rate of
hydrolysis of the arabinose, arabinose can be selectively
hydrolyzed from corn fiber arabinoxylan to provide an arabinose
enriched stream from which pure arabinose and a lower molecular
weight xylan consisting essentially of xylose can be obtained.
Thus, by using a two stage hydrolysis, the arabinose and the xylose
can be obtained in highly enriched forms without having to use
costly separation techniques such as chromatography. Such an
unexpected result provides marked economies of scale with the
processes of the invention herein and allows the separation of the
xylose and arabinose more directly from the extracted arabinoxylan
in a continuous process.
Example 33
Separation of Xylose and Ribose From Corn Fiber Arabinoxylan
[0345] A mixture of D-xylose (163 mg) and L-arabinose (102 mg) was
dissolved in a minimal amount of water (<3 mL). Methanol and
silica gel were added to the solution and the mixture was swirled
to promote mixing. The solvents were then removed under reduced
pressure. To the top of a typically prepared chromatography column
of silica gel was added the dry powder containing the mixture of
D-xylose, L-arabinose and silica gel described above. The sugars
were eluted from the column with an 8:8:1 mixture of ethyl
acetate/isopropyl alcohol/water and 1 mL samples were collected.
Tubes 19 to 25 were pooled and concentrated. Thin layer
chromatography and .sup.1H NMR of the resultant material indicated
only D-xylose was present in those fractions. Tubes 27 to 35 were
pooled and concentrated. Thin layer chromatography and .sup.1H NMR
of the resultant material indicated the presence of L-arabinose
(.about.95%) and D-xylose (.about.5%) in this fraction.
[0346] This Example illustrates that a mixture of xylose and
arabinose, such as that obtained by the method of Example 31, can
be cleanly separated by simple column chromatography over silica
gel.
Example 34
Epimerization of Arabinose to Ribose
[0347] 1.05 g of ammonium dimolybdate was added to a solution of 10
g of L-Arabinose in 45 mL of acetic acid and 10 mL of water at
50.degree. C. The reaction was stirred at 50.degree. C. for 14.8 h
before concentrating under vacuum to obtain a green oil. The oil
was taken up in water and stirred over Amberlite IRA-400 ion
exchange resin (Restek Corp., Bellefonte, Pa.) before filtering
through charcoal. The pale yellow solution was concentrated to
provide 11.2 g of an oil. The oil was taken up in hot methanol,
which resulted in the formation of crystals. The crystals were
separated by filtration, washed with cold methanol, and dried which
provided 3.68 g of white crystals which were shown to be arabinose
by .sup.1H NMR. The filtrate was concentrated in vacuo, which gave
6.86 g of an oil. Proton NMR revealed this oil to be a mixture of
L-ribose (about 24%) and L-arabinose.
[0348] This Example illustrates that arabinose can be converted to
ribose with molybdium in a solvent containing acetic acid. Thus,
arabinose obtained by hydrolysis of corn fiber arabinoxylan in
acetic acid/water can be utilized directly in the epimerization of
arabinose to ribose without intermediate isolation of the arabinose
from the acetic acid/water mixture which provides for a simple and
continuous industrial process.
Example 35
Pulse Tests for Separation of Arabinose and Xylose
[0349] To determine the feasibility of separating arabinose and
xylose using SMB chromatography, a synthetic mixture of xylose
(35%) and arabinose (20%) in water was pulsed into a 1"
ID.times.100 cm column packed with Dowex Monosphere 99 Ca/320. The
column volume was 500 mL, the pulse volume was 50 mL, the flow rate
was 9 mL min.sup.-1 and the temperature was 60.degree. C. The
results of the pulse test are presented in FIG. 12. A 0.09 bed
volume (BV) peak to peak separations was achieved indicating that
arabinose and xylose could be separated in a SMB unit.
[0350] Corn fiber arabinoxylan was hydrolyzed to a mixture of
monosaccharides as set out above. This feed was pulsed into a 1"
ID.times.100 cm column packed with Dowex Monosphere 99 Ca/320. The
column volume was 500 mL, the pulse volume was 50 mL, the flow rate
was 9 mL min.sup.-1, and the temperature was 60.degree. C. The
results of the pulse test are presented in FIG. 13. A 0.1 bed
volume (BV) peak to peak separations was achieved indicating that
the feed could be separated in a SMB unit. Because galactose and
the minor amount of glucose elude between xylose and arabinose, the
pulse test indicates that either xylose or arabinose can be highly
purified in a binary separation with the bulk of the remaining
monosaccharides going to the other component.
Example 36
Pulse Tests for Separation of Arabinose and Ribose
[0351] To determine the feasibility of separating arabinose and
ribose using SMB chromatography, a synthetic mixture of arabinose
(35%) and ribose (20%) in water was pulsed into a 1" ID.times.100
cm column packed with Dowex Monosphere 99 Ca/320. The column volume
was 500 mL, the pulse volume was 50 mL, the flow rate was 8.3 mL
min.sup.-1 and the temperature was 60.degree. C. The results of the
pulse test are presented in FIG. 14. A 0.3 bed volume (BV) peak to
peak separations was achieved indicating that arabinose and ribose
could be easily separated in a SMB unit.
Example 37
Pulse Tests for Separation of Arabinose and Xylose Using Strongly
Basic Resin in the Phosphate Form
[0352] Strongly basic resin in the phosphate form was prepared
first soaking Dowex-1 strong basic resin in the chloride form in
water. The resin was transferred to a 1 inch.times.1 meter column.
The column was rinsed with 2 bed volumes of deionized water at 8 ml
min-1 before passing 5 bed volumes of a 5% NaOH solution through
the column. The column was washed with 4 bed volumes of deionized
water before passing 5 bed volumes of a 5% H.sub.2KPO.sub.4
solution through the column. The column was then washed with 4 bed
volumes of deionized water.
[0353] A synthetic mixture of xylose (8.7%) and arabinose (5.15%)
in water was pulsed into a 1" ID.times.100 cm column packed with
the strongly basic resin in the phosphate form described above. The
column volume was 500 mL, the pulse volume was 50 mL, the flow rate
was 9 mL min.sup.-1, and the temperature was 60.degree. C. The
results of the pulse test are presented in FIG. 15. A 0.1 bed
volume (BV) peak to peak separations was achieved indicating that
arabinose and xylose could be separated in a SMB unit. A Surprising
feature of this invention is that the arabinose elutes before
xylose which is opposite to that observed with the cation resin in
the calcium form described above.
[0354] Corn fiber arabinoxylan was hydrolyzed to a mixture of
monosaccharides as set out above. This feed was pulsed into a 1"
ID.times.100 cm column packed with the strongly basic resin in the
phosphate form described above. The column volume was 500 mL, the
pulse volume was 50 mL, the flow rate was 9 mL min.sup.-1, and the
temperature was 60.degree. C. A 0.08 bed volume (BV) peak to peak
separations was achieved indicating that the feed could be
separated in a SMB unit.
Example 38
Arabinofuranosidase Catalyzed Selective Hydrolysis Arabinoxylan
[0355] A 25-mL round-bottomed flask was charged with 0.5 g of
arabinoxylan and 10 mL of 0.1 M NaOAc buffer (pH 4). The mixture
was thoroughly mixed on a vortex mixer before placing the flask
into a 50.degree. C. oil bath where the mixture was stirred for
approximately 5 minutes. The flask was capped with a rubber septum
and vented with a 20 gauge needle before adding 200 .mu.L (30
units) of A. niger arabinofuranosidase. The reaction mixture was
stirred at 50.degree. C. for approximately 64 hours before the
sample was added in small portions to 35 mL of EtOH in a centrifuge
tube with thorough mixing. The mixture was allowed to stand at room
temperature for 15 minutes before centrifuging (5 min.,
4050.times.g, 4000 rpm, 10.degree. C.). The supernatant was
decanted from the pellet which formed. The pellet was resuspended
in 25 mL of EtOH and centrifuged (5 min., 4050.times.g, 4000 rpm,
10.degree. C.). The wash was decanted from the pellet. The pellet
was dried at 50.degree. C. in vacuo. The pellet (polysaccharide
precipitate) and the supernatant (monosaccharides) were submitted
for carbohydrate analysis. The normalized carbohydrate composition
is provided in Table 25.
26TABLE 25 NORMALIZED CARBOHYDRATE COMPOSITION OF PELLET AND
SUPERNATANT AFTER TREATMENT OF ARABINOXYLAN WITH A. NIGER
ARABINOFURANOSIDASE. Sample % Glu % Xyl % Gal % Ara Pellet 6.1 61.7
9.0 23.3 Supernatant 0 0 5.5 94.5
[0356] This Example demonstrates that an arabinofuranosidase can be
used to selectively hydrolyze the arabinoxylan from corn fiber to
produce a solution highly enriched in arabinose.
Example 39
Epimerization of L-Arabinose to L-Ribose With Ammonium
Dimolybdate
[0357] To a 25 mL single neck round bottom flask equipped with a
magnetic stirrer, and condensation column was added 5 g of
L-Arabinose and 12.5 mL of distilled H.sub.2O at a pH of 2. The
reaction was heated to the desired temperature in an oil bath. Once
the reaction was clear, 1 wt. % (based on L-arabinose) of catalyst
was added and the reaction mixture was adjusted to desired pH by
addition of the appropriate amount of acid or base in distilled
H.sub.2O. The reaction was stirred for five hours at the desired
temperature. After five hours, the reaction was cooled to room
temperature and 1 g of AG-501-X8 mixed ion exchange resin was
added. This mixture was stirred for 20 minutes. The solution was
filtered through a pad of charcoal and concentrated in vacuo. The
crude mixture was analyzed on Gemini 300 NMR spectrometer (Varian,
Palo Alto, Va.) and by HPLC. The relevant reaction parameters and
results are summarized in Table 26.
27TABLE 26 EPIMERIZATION OF L-ARABINOSE TO L-RIBOSE WITH AMMONIUM
DIMOLYBDATE. % % Total % Ribose Equiv. Ribose Ribose Rib + Arb
Normalized of Temp Sample (.sup.1H) (HPLC) (HPLC) (HPLC) Catalyst
Catalyst pH (.degree. C.) 1 7.5 8.7 89.4 9.7 0.0076 Ammonium 3.5 80
Dimolybdate 2 7.6 8.4 86.6 9.7 0.0076 Ammonium 4 80 Dimolybdate 3
12.4 21.0 93.6 22.4 0.0076 Ammonium 4 90 Dimolybdate 4 17.1 16.2
89.2 18.2 0.0076 Ammonium 1 100 Dimolybdate 5 23.7 24.3.sup.1
87.0.sup.1 27.9.sup.1 0.0076 Ammonium 2 100 Dimolybdate 6 21.1 22.8
87.8 26.0 0.0076 Ammonium 2.5 100 Dimolybdate 7 20.3 22.8 92.4 24.7
0.0076 Ammonium 3 100 Dimolybdate 8 19 22.4 98.3 22.8 0.0076
Ammonium 3.5 100 Dimolybdate 9 16.6 18.3 91.1 20.2 0.0076 Ammonium
4.5 100 Dimolybdate 10 6.3 7.3 88.3 8.3 0.0076 Ammonium 6 100
Dimolybdate 11 15.9 15.1 83.5 18.1 0.0062 Sodium 2.5 100 Molybdate
12 17.4 18.4 87.7 21.0 0.0104 MoO.sub.3 2.5 100 Notes for Table 26:
.sup.1Average value for two experiments.
[0358] This Example reveals a number of interesting features of
this reaction. Of the three Mo VI catalysts examined, ammonium
dimolybdate gave the highest conversion to L-ribose under
equivalent reaction conditions (compare Samples 6, 11, 12).
Increasing the temperature at a fixed pH provides for higher
conversion of L-arabinose to L-ribose (cf. Samples 2 and 3, Samples
1 and 8). From Samples 4 through 10, it can be seen that as the pH
is decreased, the conversion to L-ribose increases with the optimum
pH being in the range of 2-2.5. However, it is worth noting that in
the pH range of 2-2.5, analysis by HPLC showed that only ca. 87% of
the mixture was L-ribose and L-arabinose indicating other side
reactions (Samples 5 and 6). At pH 3.5 (Sample 8) the side
reactions are greatly minimized but the conversion is less.
Example 40
Epimerization of L-Arabinose to L-Ribose With a Preformed
Polymolybdate Catalyst
[0359] To a 15 mL single neck round bottom flask equipped with a
magnetic stirrer and condensation column was added 6 mL of water at
a pH of 2.5 and 1 g of ammonium dimolybdate. To the flask was added
either 1 weight equivalent of boric acid or 0.5 weight equivalent
of boric acid. The flask containing the homogeneous solution was
then placed in an oil bath preheated to 100.degree. C. After
stirring for approximately 2 h, a white solid began to form. Three
hours after placing the flask in the oil bath, the reaction was
allowed to cool and the solids were removed by filtration. The
solids were washed with water and excess water was removed by
filtration but the solids were not allowed to dry. The filtrate was
concentrated in vacuo to a powder.
[0360] To a 25 mL single neck round bottom flask equipped with a
magnetic stirrer, and condensation column was added 5 g of
L-Arabinose and 12.5 mL of distilled H.sub.2O with a pH of 2. The
reaction was heated to 100.degree. C. in an oil bath. Once the
reaction was clear, 1 wt. % (based on L-arabinose) of catalyst was
added and the reaction mixture was stirred until a clear solution
was obtained. It is important to note that the solid catalyst will
not dissolve in the absence of L-arabinose. The pH of the reaction
mixture was adjusted to desired pH by addition of the appropriate
amount of acid or base in distilled H.sub.2O. The reaction was
stirred for five hours at the desired temperature. After five
hours, the reaction was cooled to room temperature and 1 g of
AG-501-X8 mixed ion exchange resin was added. This mixture was
stirred 20 minutes. The solution was filtered through a pad of
charcoal and concentrated in vacuo. The crude mixture was analyzed
on Gemini 300 NMR spectrometer and by HPLC. The relevant reaction
parameters and results are summarized in Table 27.
28TABLE 27 EPIMERIZATION OF L-ARABINOSE TO L-RIBOSE WITH PREFORMED
POLYMOLYBDATE. % % Total % Ribose Ribose Ribose Rib + Arb
Normalized Wt % Sample (.sup.1H) (HPLC) (HPLC) (HPLC) Catalyst
Catalyst pH 1.sup.1 0 NH.sub.4Mo.sub.2O.sub.5 + 2.5 eqs boric 3
acid 2.sup.2 21.1 22.8.sup. 87.8.sup. 26.0.sup. 1
NH.sub.4Mo.sub.2O.sub.5 2.5 3 8.6 nd nd nd 1 2/1
NH.sub.4Mo.sub.2O.sub.5/Boric Acid 3 (filtrate) 4 27.6 23.3.sup.3
89.5.sup.3 26.0.sup.3 1 2/1 NH.sub.4Mo.sub.2O.sub.5/Boric Acid 2.5
(solid) 5.sup.4 19.0 19.4.sup. 84.3.sup. 23.0.sup. 1 2/1
NH.sub.4Mo.sub.2O.sub.5Boric Acid 3 (filtrate) 6 21.8 22.5.sup.
88.2.sup. 25.5.sup. 1 1/1 NH.sub.4Mo.sub.2O.sub.5/Bori- c Acid 2.5
(solid) 7 1.5 3.9.sup. 100 .sup. 3.9.sup. 1 1/1
NH.sub.4Mo.sub.2O.sub.5/Boric Acid 2.5 (filtrate) 8.sup.5 22.1
21.8.sup. 81.9.sup. 26.6.sup. 1 2/1 NH.sub.4Mo.sub.2O.sub.5/Boric
Acid 2.5 (solid) 9.sup.5 23.0 19.6.sup. 75.7.sup. 25.9.sup. 1 1/1
NH.sub.4Mo.sub.2O.sub.5/Boric Acid 2.5 (solid) 10.sup.6 22.7
21.2.sup. 78.3.sup. 27.1.sup. 1 NH.sub.4Mo.sub.2O.sub.5 2.0 Notes
for Table 27: .sup.1The reaction temperature was 95.degree. C., the
pH of the reaction was 3.0, and the reaction time was 0.5 h;
.sup.2This Sample corresponds to Example 39 that does not provide
for formation of polymolybdate; .sup.3Average value for two
experiments; .sup.4The catalyst was prepared at a pH of 5 rather
than the standard pH of 2.5 which does not lead to the formation of
a solid catalyst; .sup.5The catalyst was dried in vacuo to remove
water prior to the epimerization reaction; .sup.6The
NH.sub.4Mo.sub.2O.sub.5 was treated as described above with
NH.sub.4Mo.sub.2O.sub.5 + boric acid.
[0361] Sample 1 is a comparative example (see U.S. Pat. No.
4,602,086, Example 1). In this example, the Mo VI catalyst, large
excess of boric acid, and L-arabinose were combined and no attempt
was made to isolate a solid polymolybdate catalyst like the present
invention. Moreover, practice of the invention of Sample 1 does not
provide for formation of L-ribose. Only L-arabinose and the methyl
glycoside are obtained.
[0362] Sample 2 corresponds to the method disclosed in Example
39which does not require formation of a solid catalyst. Although
the normalized L-ribose values from HPLC are similar to those
obtained using the solid catalyst described in this example
(Samples 4 and 6), the sum of the L-ribose and L-arabinose values
indicates that more side reactions occur with the catalyst of
Sample 2 (12.2%) versus those of Sample 4 (10.5%) or Sample 6
(11.8%).
[0363] Samples 6 and 7 provide comparisons of the activity of solid
catalyst obtained in this invention and the activity of the
catalyst obtained after concentration of the filtrate to a powder.
These Samples show that the active catalyst is the solid catalyst.
This is particularly evident from Sample 5. In this example, the
catalyst was prepared at a pH of 5 rather than the standard pH of
2.5 which does not lead to the formation of a solid catalyst. In
this case, the active catalyst is found in the filtrate and it is
not nearly as active as the equivalent solid catalyst (cf. Samples
4 and 5).
[0364] Samples 8 and 9 show that the solid catalyst should not be
dried. That is, they should remain in the hydrated form. The
catalyst of Sample 8 results from taking a small fraction of the
catalyst of Sample 4 and drying it to remove all of the water.
Similarly, the catalyst of Sample 9 results from taking a small
fraction of the catalyst of Sample 6 and drying it to remove all of
the water. Although the normalized L-ribose values from HPLC are
equivalent, the sum of the L-ribose and L-arabinose values
indicates that many more side reactions are occurring with
extensively dried catalysts.
[0365] Lastly, the catalyst of Sample 10 was formed as described
above except that there was no boric acid added. Although the
normalized L-ribose values are equivalent to those obtained from
the NH.sub.4Mo.sub.2O.sub.5/boric acid polymolybdate (compare
Sample 10 to Samples 4 and 6), the sum of the L-ribose and
L-arabinose values indicates that many more side reactions are
occurring with the catalyst of Sample 10 relative to Samples 4 and
6. That is, the catalysts of Samples 4 and 6 provide for the best
combination of conversion of L-arabinose to L-ribose with minimal
side reactions.
[0366] The invention has been described in detail with particular
reference to preferred embodiments thereof, but it will be
understood that variations and modifications can be effected
without departing from the scope and spirit of the invention.
* * * * *