U.S. patent application number 14/546250 was filed with the patent office on 2015-05-28 for high force and high stress destructuring for starch biomass processing.
The applicant listed for this patent is E I DU PONT DE NEMOURS AND COMPANY. Invention is credited to KATHLEEN A CLARKSON, F Glenn Gallagher, Aaron Perelman, Luis Fernando Romero Millan, Vivek Sharma, Jayarama K. Shetty, Daniel A. Slanac, Paula Johanna Maria Teunissen.
Application Number | 20150147786 14/546250 |
Document ID | / |
Family ID | 52014430 |
Filed Date | 2015-05-28 |
United States Patent
Application |
20150147786 |
Kind Code |
A1 |
CLARKSON; KATHLEEN A ; et
al. |
May 28, 2015 |
HIGH FORCE AND HIGH STRESS DESTRUCTURING FOR STARCH BIOMASS
PROCESSING
Abstract
A process for mechanical destructuring of starch-based biomass
was developed that makes use of a short application of high
compression, impact, and shearing forces. The biomass may be
destructured using a specific energy input that is less than 40% of
the total combustible energy of the biomass. The destructured
starch-based biomass, with or without saccharification and/or
in-feed glycosyl hydrolase enzymes, may be used in feed
applications. The destructured starch-based may saccharified to
produce syrups and fermentable sugars, and for production of
products including ethanol using a biocatalyst.
Inventors: |
CLARKSON; KATHLEEN A; (San
Francisco, CA) ; Gallagher; F Glenn; (Wilmington,
DE) ; Perelman; Aaron; (Wilmington, DE) ;
Romero Millan; Luis Fernando; (Swindon, GB) ; Sharma;
Vivek; (Fremont, CA) ; Shetty; Jayarama K.;
(Pleasonton, CA) ; Slanac; Daniel A.; (Greenville,
DE) ; Teunissen; Paula Johanna Maria; (Saratoga,
CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
E I DU PONT DE NEMOURS AND COMPANY |
Wilmington |
DE |
US |
|
|
Family ID: |
52014430 |
Appl. No.: |
14/546250 |
Filed: |
November 18, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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62014313 |
Jun 19, 2014 |
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61908158 |
Nov 24, 2013 |
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Current U.S.
Class: |
435/95 ; 127/29;
127/67; 426/2; 426/61; 426/623; 426/72; 435/106; 435/110; 435/115;
435/125; 435/126; 435/134; 435/136; 435/137; 435/139; 435/144;
435/145; 435/158; 435/160; 435/162; 435/167; 435/96; 435/99 |
Current CPC
Class: |
A61K 31/717 20130101;
C12P 19/20 20130101; C12P 2201/00 20130101; C08H 8/00 20130101;
A23K 10/30 20160501; C13K 1/02 20130101; Y02E 50/10 20130101; Y02P
60/877 20151101; C12P 19/02 20130101; A61K 38/47 20130101; C08B
30/02 20130101; A23K 20/189 20160501; Y02P 60/87 20151101; C12P
7/14 20130101; C13K 13/002 20130101; C12Y 302/01004 20130101; C08B
30/12 20130101; Y02E 50/16 20130101; C12P 19/14 20130101; C12P 7/10
20130101 |
Class at
Publication: |
435/95 ; 435/99;
435/96; 435/162; 435/136; 435/144; 435/139; 435/145; 435/110;
435/137; 435/125; 435/126; 435/134; 435/160; 435/106; 435/115;
435/158; 435/167; 127/67; 127/29; 426/623; 426/61; 426/72;
426/2 |
International
Class: |
C08B 30/02 20060101
C08B030/02; C12P 19/02 20060101 C12P019/02; A23K 1/165 20060101
A23K001/165; C12P 7/14 20060101 C12P007/14; C08B 30/12 20060101
C08B030/12; A23K 1/14 20060101 A23K001/14; C12P 19/14 20060101
C12P019/14; C12P 19/20 20060101 C12P019/20 |
Claims
1. A process for producing destructured starch-based biomass
comprising: a) providing a starch-based biomass; and b) applying to
the biomass of (a) at least one set of compression and impact
forces of at least 5,000 N combined with shearing forces; wherein
contact stress of greater than 5,000 psi is applied to the biomass
and wherein a destructured starch-based biomass is produced.
2. A process for producing destructured starch-based biomass
comprising: a) providing a portion of starch-based biomass; and b)
applying to the biomass of (a) at least one set of compression and
impact forces of at least 1,500 N combined with shearing forces,
and contact stress of greater than 5,000 psi; wherein specific
energy input in the process is less than 40% of the total
combustible energy of the portion of biomass being treated and
wherein a destructured starch-based biomass is produced.
3. The process of claim 2 wherein the compression and impact forces
are at least 3,000 N.
4. The process of claim 1 or 2 wherein the stress is greater than
about 8,000 psi.
5. The process of claim 4 wherein the stress is greater than about
13,000 psi.
6. The process of claim 1 wherein the forces are imparted using a
non-vibratory apparatus which does not contain free flowing
media.
7. The process of claim 1 or 2 wherein the forces are imparted by
centrifugal motion, hydraulics, or springs.
8. The process of claim 7 wherein the forces are imparted by
centrifugal motion and the Radii Ratio is less than 0.5.
9. The process of claim 8 wherein the forces are imparted by
centrifugal motion and the Radii Ratio is less than 0.4.
10. The process of claim 9 wherein the forces are imparted by
centrifugal motion and the Radii Ratio is about 0.33.
11. The process of claim 1 or 2 wherein the starch-based biomass is
selected from the group consisting of grains and starchy roots or
tubors.
12. The process of claim 11 wherein grains is selected from corn,
rice, wheat, barley, sorghum, millet, rye, milo, triticale, and
mixtures thereof.
13. The process of claim 11 wherein starchy roots are roots
selected from potato, sweet potato, yam, taro, carrot, turnip,
cassava, and mixtures thereof.
14. The process of claim 1 or 2 wherein the destructured
starch-based biomass has crystallinity that is reduced by at least
about 25% as compared to the starch-based biomass provided in
(a).
15. The process of claim 1 or 2 wherein each individual application
of force occurs in less than ten milliseconds.
16. The process of claim 1 or 2 wherein compression and impact
forces applied to the biomass are applied by smooth surfaces.
17. The process of claim 1 wherein G-force is applied at less than
10 G.
18. The process of claim 1 or 2 wherein at least one set of
compression and impact forces is at least about 10,000 newtons
19. The process of claim 18 wherein at least one set of compression
and impact forces is at least about 50,000 newtons
20. The process of claim 19 wherein at least one set of compression
and impact forces is at least about 100,000 newtons
21. The process of claim 7 wherein the compression and impact
forces are imparted by centrifugal motion by moving an object with
a mass of at least 100 kilograms.
22. The process of claim 1 or 2 further comprising contacting the
destructured starch-based biomass with at least one
saccharification enzyme under suitable conditions for
saccharification wherein sugars are produced.
23. A destructured starch-based biomass obtainable by the process
of claim 1 or 2 comprising starch having at least 25% reduction in
crystallinity as compared to provided starch-based biomass.
24. The destructured starch-based biomass of claim 23 having a
processed energy equivalency of greater than 60%.
25. The destructured starch-based biomass of claim 23 further
comprising a destructured cellulosic biomass component.
26. The composition of claim 23 further comprising free oil.
27. A feed additive composition comprising a destructured
starch-based biomass obtainable (e.g. obtained) by the process of
any one of claims 1 to 22.
28. A feed additive composition comprising a destructured
starch-based biomass obtainable (e.g. obtained) by the process of
any one of claims 1 to 22 in combination with at least one in-feed
enzyme.
29. A feed additive kit comprising a first container comprising a
destructured starch-based biomass obtainable (e.g. obtained) by the
process of any one of claims 1 to 22 and a second container
comprising at least one in-feed enzyme, and instructions for
co-administering same.
30. A feed or feedstuff comprising a feed additive composition
according to claim 27 or claim 28.
31. A premix comprising a feed additive composition according to
claim 27 or claim 28, and at least one mineral and/or at least one
vitamin.
32. A method for improving a biophysical characteristic of an
animal which method comprises administering to an animal a feed
additive composition according to claim 27 or claim 28 or a feed
according to claim 30 or a premix according to claim 31.
33. Use of a feed additive composition according to claim 27 or
claim 28 or a feed according to claim 30 or a premix according to
claim 31 for improving a biophysical characteristic of an
animal.
34. A method of preparing a feedstuff comprising contacting a feed
component with a feed additive composition according to claim 27 or
claim 28.
35. A method of saccharifying a composition comprising destructured
starch-based biomass to produce a composition comprising glucose,
wherein said method comprises: (i) contacting a destructured
starch-based biomass composition with a glucoamylase; and (ii)
saccharifying the composition to produce said glucose
composition.
36. The method of claim 35, wherein the destructured starch-based
biomass is from a process of any one of claim 1-22.
37. The method of claim 35 or claim 36, wherein the glucose
composition is produced without a liquefaction step.
38. The method of any one of claims 35-37, wherein the glucose
composition has a DP1 concentration higher than would result when
saccharifying a whole ground corn liquefact composition in step (i)
in place of the destructured starch-based biomass composition;
wherein the higher DP1 concentration is selected from the list
consisting of (a) at least 0.1% higher DP1; (b) at least 0.2%
higher DP1; (c) at least 0.4% higher DP1; and (d) at least 0.5%
higher DP1.
39. The method of any one of claims 35-38, wherein the glucose
composition has a DP2 concentration lower than would result when
saccharifying a whole ground corn liquefact composition in step (i)
in place of the destructured starch-based biomass composition;
wherein DP2 concentration at least 0.1% lower.
40. A method of saccharifying a composition comprising destructured
starch-based biomass to produce a composition comprising maltose,
wherein said method comprises: (i) contacting a destructured
starch-based biomass composition with a maltogenic alpha-amylase;
and (ii) saccharifying the composition to produce said maltose
composition.
41. The method of claim 40, wherein the destructured starch-based
biomass composition is from a process of any one of claim 1-22.
42. The method of claim 40 or claim 41, wherein the maltose
composition is produced without one or both of a liquefaction step
and addition of an additional alpha-amylase.
43. The method of any one of claims 40-42, wherein the maltose
composition has a DP2 concentration higher than would result when
saccharifying a whole ground corn liquefact composition in step (i)
in place of the destructured starch-based biomass composition;
wherein the higher DP2 concentration is at least 0.1% higher.
44. A method of saccharifying a composition comprising destructured
starch-based biomass to produce a composition comprising
maltoriose, wherein said method comprises: (i) contacting a
destructured starch-based biomass composition with a beta-amylase
and optionally a pullulanase; and (ii) saccharifying the
composition to produce said maltotriose composition.
45. The method of claim 44, wherein the destructured starch-based
biomass composition is from a process of any one of claim 1-22.
46. The method of claim 44 or claim 45, wherein the maltotriose
composition is produced without one or both of a liquefaction step
and addition of an additional alpha-amylase.
47. The method of any one of claims 44-46, wherein the maltotriose
composition has lower DP1 and DP2 concentrations than would result
when saccharifying a whole ground corn liquefact composition in
step (i) in place of the destructured starch-based biomass
composition; wherein the DP1 and DP2 concentrations are at least
0.1% lower.
48. A method of saccharifying a composition comprising destructured
starch-based biomass to produce a composition comprising
maltotetraose, wherein said method comprises: (i) contacting a
destructured starch-based biomass composition with a DP4 producing
alpha-amylase and optionally a pullulanase; and (ii) saccharifying
the composition to produce said maltotetraose composition.
49. The method of claim 48, wherein the destructured starch-based
biomass composition is from a process of any one of claim 1-22.
50. The method of claim 48 or claim 49, wherein the maltotetraose
composition is produced without one or both of a liquefaction step
and addition of an additional alpha-amylase.
51. The method of any one of claims 48-59, wherein the
maltotetraose composition has a DP4 concentration higher than would
result when saccharifying a whole ground corn liquefact composition
in step (i) in place of the destructured starch-based biomass
composition; wherein the higher DP4 concentration is selected from
the list consisting of (a) at least 0.1% higher DP4; (b) at least
0.2% higher DP4; (c) at least 0.4% higher DP4; and (d) at least
0.5% higher DP4.
52. A method of saccharifying a composition comprising destructured
starch-based biomass to produce a composition comprising
maltopentaose, wherein said method comprises: (i) contacting a
destructured starch-based biomass composition with a DP5 producing
alpha-amylase and optionally a pullulanase; and (ii) saccharifying
the composition to produce said maltopentaose composition.
53. The method of claim 52, wherein the destructured starch-based
biomass composition is from a process of any one of claim 1-22.
54. The method of claim 52 or claim 53, wherein the maltopentaose
composition is produced without one or both of a liquefaction step
and addition of an additional alpha-amylase.
55. The method of any one of claims 52-54, wherein the
maltopentaose composition has a DP5 concentration higher than would
result when saccharifying a whole ground corn liquefact composition
in step (i) in place of the destructured starch-based biomass
composition; wherein the higher DP5 concentration is selected from
the list consisting of (a) at least 0.1% higher DP5; (b) at least
0.2% higher DP5; (c) at least 0.4% higher DP5; (d) at least 0.5%
higher DP5; (e) at least 1.0% higher DP5; (f) at least 1.5% higher
DP5; and (g) at least 2.0% higher DP5.
56. The method of any one of claims 35-55, wherein saccharifying is
conducted at a temperature range of about 30.degree. C. to about
65.degree. C.
57. The method of claim 56, wherein said temperature range is
47.degree. C. to 60.degree. C.
58. The method of any one of claims 35-57, further comprising
fermenting the produced composition to produce an End of
Fermentation (EOF) product.
59. The method of claim 58, wherein said fermentation is a
simultaneous saccharification and fermentation (SSF) reaction.
60. The method of claim 58 or 59, wherein said fermentation is
conducted for 24-70 hours at pH 2-8 and in a temperature range of
25.degree. C.-70.degree. C.
62. The method of any one of claims 58-60, wherein the EOF product
comprises ethanol.
63. The method of any one of claims 58-61, wherein the EOF product
comprises 8%-18% (v/v) ethanol.
64. The method of any one of claims 58-60, wherein the EOF product
comprises a metabolite.
65. The method of claim 64, wherein the metabolite is an organic
acid, citric acid, lactic acid, succinic acid, monosodium
glutamate, gluconic acid, sodium gluconate, calcium gluconate,
potassium gluconate, glucono delta-lactone, sodium erythorbate,
omega 3 fatty acid, butanol, an amino acid, lysine, itaconic acid,
1,3-propanediol, or isoprene.
66. The method of any one of claims 35-65, further comprising
adding in step (i) or step (ii) an additional glucoamylase,
hexokinase, xylanase, glucose isomerase, xylose isomerase,
phosphatase, phytase, protease, pullulanase, alpha-amylase,
beta-amylase, protease, cellulase, hemicellulase, lipase, cutinase,
trehalase, isoamylase, redox enzyme, esterase, transferase,
pectinase, alpha-glucosidase, beta-glucosidase, lyase, hydrolase,
or a combination thereof.
67. A method of producing alcohol from a destructured starch-based
biomass substrate by simultaneous saccharification and fermentation
(SSF) comprising, (i) contacting a slurry comprising destructured
starch-based biomass with an alpha-amylase, a glucoamylase, a yeast
and optionally an acid stable protease, at a temperature below the
starch gelatinization temperature of the destructured starch-based
biomass to produce oligosaccharides fermentable by the yeast; (ii)
fermenting the oligosaccharides to produce alcohol.
68. The method of claim 67, wherein the destructured starch-based
biomass composition is from a process of any one of claim 1-22.
69. The method of claim 67 or claim 68, wherein the initial
percentage of alcohol production over the initial phase of SSF is a
higher percentage than would result when using a milled corn flour
or whole ground corn composition in step (i) in place of the
destructured starch-based biomass composition.
70. The method of any one of claims 67-69, wherein the initial
phase of SSF is the first 15 minutes of SSF.
71. The method of claim 69 or claim 70, wherein the higher
percentage of alcohol resulting in the initial phase of SSF is a
fold higher % selected from the list consisting of (a) at least 2
fold higher % vs. corn flour; and (b) at least 4 fold higher % vs.
whole ground corn.
72. The method of claim 67 or claim 68, wherein the end of SSF
total alcohol yield requires 3 fold less glucoamylase dosage as
when using a milled corn flour or whole ground corn composition in
step (i) in place of the destructured starch-based biomass
composition.
73. The method of claim 67 or claim 68, wherein up to 10 fold less
glucoamylase is required to produce a comparable end of SSF alcohol
yield for destructured starch-based biomass composition than would
be required for corn flour.
74. The method of any one of claims 67-73, wherein the alcohol
produced is ethanol.
75. The method of any one of claims 35-70, wherein the destructured
starch-based biomass composition is selected from the group
consisting of corn, barley, cassava, rice, rye, milo, millet,
sorghum, triticale, wheat and any combination thereof.
76. The method of any one of claims 35-70, wherein the destructured
starch-based biomass composition is selected from the group
consisting of corn, cassava, sorghum, wheat and any combination
thereof.
77. The method of any one of claims 35-70, wherein the destructured
starch-based biomass composition is corn.
78. The method of any one of claims 35-70, wherein the destructured
starch-based biomass comprises a composition obtained from
processed starch-based biomass.
Description
[0001] This application claims the benefit of U.S. Provisional
Applications 61/908,158 and 62/014,313, filed Nov. 24, 2013 and
Jun. 19, 2014, respectively, now pending, which are incorporated in
their entirety herein by reference.
FIELD OF THE INVENTION
[0002] Processes for destructuring biomass that is high in starch
content are provided. Specifically, starch-based biomass is
processed using high compression, impact, and shearing forces
thereby producing a destructured starch-based biomass product that
is digestible and from which sugars can be obtained.
BACKGROUND OF THE INVENTION
[0003] In a typical bio-ethanol production process corn kernels are
used as a source of starch to provide sugars utilized by a
biocatalyst for fermentative production of ethanol. Typically the
starch in corn kernels is made available by dry grinding, where the
corn is ground into flour which is prepared in a slurry of
insoluble starch granules, which is then heat treated at
temperatures between 80.degree. C. and 100.degree. C. in the
presence of thermostable alpha-amylases producing low viscosity
mash in a process called liquefaction. During liquefaction, the
heat causes the starch granules to swell and burst releasing
long-chained starch molecules and causing a dramatic increase in
viscosity, which is reduced by enzymatic degradation of the
long-chained starch molecules by alpha-amylase into dextrins, which
are shorter chains of glucose. Saccharification enzymes, typically
various amylases, glucosidases, and/or glucanases, are then added
to break down the dextrins producing sugars that can be fermented
by a biocatalyst. Fermentation may be a separate step, or combined
with saccharification in a simultaneous saccharification and
fermentation (SSF) step. Other grains such as wheat, barley,
sorghum, millet, and rye, as well as tubers such as cassava and
potato may provide starch in this manner as well.
[0004] No-cook processes for saccharification of corn have been
used as in U.S. Pat. No. 7,303,899, but typically much higher
amounts of enzymes are required than when using a process that
includes a heating step.
[0005] There remains a need for an improved process for treating
sources of starch-based biomass to provide material that is readily
digestible, and for simplified processes that make use of the
material.
SUMMARY OF THE INVENTION
[0006] The invention provides a process for treating starch-based
biomass by which the starch and other components of the biomass are
made available for further processing.
[0007] Accordingly, the invention provides a process for producing
destructured starch-based biomass comprising: [0008] a) providing a
starch-based biomass; and [0009] b) applying to the biomass of (a)
at least one set of compression and impact forces of at least 5,000
N combined with shearing forces; [0010] wherein contact stress of
greater than 5,000 psi is applied to the biomass and wherein a
destructured starch-based biomass is produced.
[0011] In another aspect the invention provides a process for
producing destructured starch-based biomass comprising: [0012] a)
providing a portion of starch-based biomass; and [0013] b) applying
to the biomass of (a) at least one set of compression and impact
forces of at least 1,500 N combined with shearing forces, and
contact stress of greater than 5,000 psi; [0014] wherein specific
energy input in the process is less than 40% of the total
combustible energy of the portion of biomass being treated and
wherein a destructured starch-based biomass is produced.
[0015] In another aspect the invention provides a destructured
starch-based biomass obtainable by the above process comprising
starch having at least 25% reduction in crystallinity as compared
to provided starch-based biomass.
[0016] In other aspects the invention provides a feed additive
composition, a feed additive kit, a feed or feedstuff, and a premix
comprising the destructured starch-based biomass.
[0017] In other aspects the invention provides a method for
improving a biophysical characteristic of an animal which method
comprises administering to an animal said feed additive
composition, use of said feed additive composition for improving a
biophysical characteristic of an animal, and a method of preparing
a feedstuff comprising contacting a feed component with said feed
additive composition.
[0018] In yet another aspect the invention provides a method of
saccharifying a composition comprising destructured starch-based
biomass to produce a composition comprising glucose, wherein said
method comprises: [0019] (i) contacting a destructured starch-based
biomass composition with a glucoamylase; and [0020] (ii)
saccharifying the composition to produce said glucose
composition.
[0021] In yet another aspect the invention provides a method of
saccharifying a composition comprising destructured starch-based
biomass to produce a composition comprising maltose, wherein said
method comprises: [0022] (i) contacting a destructured starch-based
biomass composition with a maltogenic alpha-amylase; and [0023]
(ii) saccharifying the composition to produce said maltose
composition.
[0024] In yet another aspect the invention provides a method of
saccharifying a composition comprising destructured starch-based
biomass to produce a composition comprising maltotriose, wherein
said method comprises: [0025] (i) contacting a destructured
starch-based biomass composition with a beta-amylase and optionally
a pullulanase; and [0026] (ii) saccharifying the composition to
produce said maltotriose composition.
[0027] In yet another aspect the invention provides a method of
saccharifying a composition comprising destructured starch-based
biomass to produce a composition comprising maltotetraose, wherein
said method comprises: [0028] (i) contacting a destructured
starch-based biomass composition with a DP4 producing alpha-amylase
and optionally a pullulanase; and [0029] (ii) saccharifying the
composition to produce said maltotetraose composition.
[0030] In yet another aspect the invention provides a method of
saccharifying a composition comprising destructured starch-based
biomass to produce a composition comprising maltopentaose, wherein
said method comprises: [0031] (i) contacting a destructured
starch-based biomass composition with a DP5 producing alpha-amylase
and optionally a pullulanase; and [0032] (ii) saccharifying the
composition to produce said maltopentaose composition.
[0033] In yet another aspect the invention provides a method of
producing alcohol from destructured starch-based biomass by
simultaneous saccharification and fermentation (SSF) comprising,
[0034] (i) contacting a slurry comprising destructured starch-based
biomass with an alpha-amylase, a glucoamylase, a yeast and
optionally an acid stable protease, at a temperature below the
starch gelatinization temperature of the destructured starch-based
biomass to produce oligosaccharides fermentable by the yeast;
[0035] (ii) fermenting the oligosaccharides to produce alcohol.
BRIEF DESCRIPTION OF THE DRAWINGS
[0036] FIG. 1 is a diagram of an apparatus for imparting
compression and impact forces using centrifugal motion.
[0037] FIG. 2A is a diagram of a side view of a ring-roller mill
that employs centrifugal force between revolving milling media and
a grinding surface.
[0038] FIG. 2B is a diagram of a top view of a ring-roller mill
that employs centrifugal force between revolving milling media and
a grinding surface.
[0039] FIG. 3A shows a graph of particle size distribution (PSD)
for ring and puck milled whole corn kernels.
[0040] FIG. 3B shows a graph of PSD for hammer milled whole corn
kernels.
[0041] FIG. 4 shows a graph of XRD Spectra for whole corn kernels
either hammer milled (corn flour; top curve) or ring and puck
milled (bottom curve).
[0042] FIG. 5A shows a graph of PSD for ring and puck milled whole
white wheat kernels.
[0043] FIG. 5B shows a graph of XRD Spectra for whole white wheat
kernels either crushed with a mortar and pestle (top curve) or ring
and puck milled (bottom curve).
[0044] FIG. 6A shows a graph of PSD for ring puck milled whole
sorghum.
[0045] FIG. 6B shows a graph of XRD Spectra for whole sorghum
either crushed with a mortar and pestle (top curve) or ring and
puck milled (bottom curve).
[0046] FIG. 7A shows a graph of PSD for ring and puck milled whole
brown rice.
[0047] FIG. 7B shows a graph of XRD Spectra for whole brown rice
either crushed with a mortar and pestle (top curve) or ring and
puck milled (bottom curve).
[0048] FIG. 8A shows a graph of PSD for ring and puck milled dried
cassava.
[0049] FIG. 8B shows a graph of XRD Spectra for dried cassava
either crushed with a mortar and pestle (top curve) or ring and
puck milled (bottom curve).
[0050] FIG. 9 shows a graph of XRD spectra of corn kernels that
were ring and puck milled for various times.
DETAILED DESCRIPTION
[0051] The invention relates to a process for pretreating
starch-based biomass with high force and stress wherein a
destructured starch-based biomass product is made. The destructured
biomass product may be saccharified producing sugars which may be
used in production of desired target products, such as through
fermentation using a biocatalyst, or by reacting with a biochemical
(enzyme) or chemical catalyst.
[0052] Alternatively the destructured starch-based biomass product
or saccharified destructed biomass (also referred to herein as
starch-based biomass hydrolysate) may be used as an animal feed or
as a feed additive composition. The destructured starch-based
biomass product or the saccharified destructured starch-based
biomass product (e.g the starch-based biomass hydrolysate) may be
admixed or combined with at least one in-feed enzyme prior to
feeding to an animal.
[0053] The following definitions and abbreviations are to be used
for the interpretation of the claims and the specification.
[0054] As used herein, the terms "comprises," "comprising,"
"includes," "including," "has," "having," "contains" or
"containing," or any other variation thereof, are intended to cover
a non-exclusive inclusion. For example, a composition, a mixture,
process, method, article, or apparatus that comprises a list of
elements is not necessarily limited to only those elements but may
include other elements not expressly listed or inherent to such
composition, mixture, process, method, article, or apparatus.
Further, unless expressly stated to the contrary, "or" refers to an
inclusive or and not to an exclusive or. For example, a condition A
or B is satisfied by any one of the following: A is true (or
present) and B is false (or not present), A is false (or not
present) and B is true (or present), and both A and B are true (or
present).
[0055] Also, the indefinite articles "a" and "an" preceding an
element or component of the invention are intended to be
nonrestrictive regarding the number of instances (i.e. occurrences)
of the element or component. Therefore "a" or "an" should be read
to include one or at least one, and the singular word form of the
element or component also includes the plural unless the number is
obviously meant to be singular.
[0056] The term "invention" or "present invention" as used herein
is a non-limiting term and is not intended to refer to any single
embodiment of the particular invention but encompasses all possible
embodiments as described in the specification and the claims.
[0057] As used herein, the term "about" modifying the quantity of
an ingredient or reactant of the invention employed refers to
variation in the numerical quantity that can occur, for example,
through typical measuring and liquid handling procedures used for
making concentrates or use solutions in the real world; through
inadvertent error in these procedures; through differences in the
manufacture, source, or purity of the ingredients employed to make
the compositions or carry out the methods; and the like. The term
"about" also encompasses amounts that differ due to different
equilibrium conditions for a composition resulting from a
particular initial mixture. Whether or not modified by the term
"about", the claims include equivalents to the quantities. In one
embodiment, the term "about" means within 10% of the reported
numerical value, preferably within 5% of the reported numerical
value.
[0058] The term "fermentable sugar" refers to oligosaccharides and
monosaccharides that can be used as a carbon source by a
microorganism in a fermentation process.
[0059] The term "lignocellulosic" describes to a composition
comprising both lignin and at least 10 wt % cellulose.
Lignocellulosic material may also comprise hemicellulose. Examples
of lignocellulosic biomass include, but are not limited to, corn
cobs, crop residues such as corn husks, corn stover, corn grain
fiber, grasses, wheat, wheat straw, barley, barley straw, hay, rice
straw, switchgrass, waste paper, sugar cane bagasse, sorghum, sweet
sorghum, soy, components obtained from milling of grains or from
using grains in production processes (such as corn fiber fraction,
DDGS: dried distillers grains with solubles), trees, branches,
roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables,
fruits, flowers, and animal manure or a combination thereof.
[0060] The term "cellulosic" describes a composition comprising at
least 10 wt % cellulose or cellulose plus hemicellulose. Cellulosic
material may also include lignin. Thus lignocellulosic biomass is a
type of cellulosic biomass. Cellulosic also refers to compositions
enriched in cellulose and/or hemicellulose obtained from processing
of grains, including but not limited to grain fractions (for
example a fiber fraction), by-products (for example wet cake,
distillers dry grains (DDG) or distillers dry grains with solubles
(DDGS)), biomass post any liquefaction, saccharification,
fermentation, SSF and/or other process or treatment.
[0061] The term "starch-based biomass" refers to a biomaterial that
is at least about 10% starch in composition by weight. Examples of
starch-based biomass include grains such as corn, wheat, barley,
rye, millet, milo, sorghum, sweet sorghum, and rice, as well as
starch storage root biomass such as potato, sweet potato, yam,
taro, carrot, turnip, triticale, sago and cassava. Starch-based
biomass may in addition contain other components such as protein,
cellulose, hemicellulose, and lignin. "Starch-based biomass" also
refers to compositions obtained from processing of starch-based
biomass wherein the processed starch-based biomass is at least
about 1% starch in composition by weight. For example, starch-based
biomass obtained from processing of grains may include grain
fractions and by-products such as fiber fractions, endosperm
fractions, wet cake, distillers dry grains (DDG) or distillers dry
grains with solubles (DDGS)), and biomass post any liquefaction,
saccharification, fermentation, SSF process and/or other process or
treatment of starch-based biomass.
[0062] The term "starchy root" refers to starch storage root
biomass.
[0063] The term "saccharification" refers to the production of
sugars from polysaccharides. Saccharification may be by any method
including enzymatic digestion and chemical treatment.
[0064] The term "destructured" refers to a biomass that has at
least a 25% reduction in crystallinity that is measured as
described in General Methods herein.
[0065] As used herein the term "compression force" refers to the
application of inward ("pushing") forces to different points on a
material or structure, that is, forces with no net sum or torque
directed so as to reduce its size in one or more directions.
Compression force is applied between two surfaces where equal and
opposite force vectors are generated normal to the curvature of the
surfaces.
[0066] As used herein an "impact force" is a high force or shock
applied over a short time period when two or more bodies collide.
For example, when a compressive force is applied over a short
period of time, this is considered as an impact force. As used
herein, the impact force is a force resulting from a fast
compression between surfaces. It is a mode of applying an impact
force that is different from a mode where force is generated by
only one surface effectively applying a force on a material (e.g. a
swinging hammer hitting material in a hammer mill). Hammer milling
or jet milling are types of milling that are used herein for
comparative purposes and do not result in destructured cellulosic
biomass in accordance with the present invention. These are used as
control treatments in the examples referred to herein.
[0067] As used herein the term "milling media" refers to any
grinding surface inside the confines of a mill's grinding chamber,
that does not include the chamber itself, including but not limited
to balls in a ball mill, rods in a rod mill, the rollers in a
ring-roller mill employing centrifugal force, the rollers in a
ring-roller mill employing spring and/or hydraulic force, rings and
puck in a puck mill, and gears in a gear-type grinding media
mill.
[0068] The term "G-force" as used herein refers to the ratio of the
actual acceleration to the gravitational acceleration, where the
gravitational acceleration is .about.9.8 m/s.sup.2. For example, if
the acceleration of the milling media is calculated to be .about.60
m/s.sup.2, then the corresponding "G-force" is .about.6 G.
[0069] "Enzyme consortium" or "saccharification enzyme consortium"
is a collection of enzymes, usually secreted by microorganisms,
which in the present case will typically contain one or more
amylases, glucoamylases, cellulases, xylanases, glycosidases,
ligninases, other hemicellulases, esterases, and other glycosyl
hydrolysas. In addition, other enzymes may be included in an enzyme
consortium to enhance access to the various carbohydrates present,
help stabilize one or more enzymes, improve saccharification and/or
SSF process, including but not limited to proteases, cutinases,
lipases, phytases, and transferases.
[0070] The term "target compound" refers to any product that is
produced by a microbial production host cell in a fermentation.
Target compounds may be the result of genetically engineered
enzymatic pathways in host cells or may be produced by endogenous
pathways. In addition, target compounds may be produced via
chemical reaction with or without a chemical catalyst. Typical
target compounds include but are not limited to acids, alcohols,
alkanes, alkenes, aromatics, aldehydes, ketones, biopolymers,
proteins, peptides, amino acids, vitamins, antibiotics, and
pharmaceuticals.
[0071] The term "coherent domain size" refers to the average
distance (length) over which crystalline order is observed in
cellulose, that is, the cellulose crystalline size that is free of
structural defects.
[0072] The term "crystalline fraction" or "crystallinity fraction"
refers to volume ratio of the crystalline portion of cellulose to
the total volume of cellulose (including both amorphous and
crystalline regions).
[0073] The term "yield" with respect to sugars refers to the amount
obtained as a percentage of the total calculated yield.
[0074] The term "processed energy equivalency" refers to an energy
percentage that is determined by the ratio of the difference of the
combustible energy of the input material minus the energy used in
processing of the material to the combustible energy of the input
material.
[0075] The phrase "simultaneous saccharification and fermentation"
or "SSF" refers to a process in the production of biochemicals in
which a microbial organism, such as an ethanologenic microorganism,
and at least one enzyme, such as one or more glucoamylases, are
present during the same process step. SSF includes the
contemporaneous hydrolysis of starch substrates to saccharides,
including glucose, and the fermentation of the saccharides into
alcohol or other biochemical or biomaterial in the same reactor
vessel.
[0076] The term "liquefaction" refers to a stage in starch
conversion in which gelatinized starch is hydrolyzed to give low
molecular weight soluble dextrins.
Pretreatment using High Force and High Stress Milling
[0077] In the present process, starch-based biomass is destructured
using mechanical forces in preparation for saccharification or use
in animal feed. In addition, biomass derived from starch-based
biomass processing, which may contain cellulosic biomass (such as
corn grain fiber) and/or contain starch, is treated using
mechanical forces for destructuring. The present process performs
mechanical destructuring of the starch-based biomass such that it
produces a destructured biomass product. The present mechanical
destructuring is effective with a short period of application for
the required power, thereby providing a process that requires
relatively low energy input in support of a commercially effective
starch-based biomass pretreatment process.
[0078] Starch-based biomass refers to any biomaterial that contains
at least about 10% starch by weight, as well as compositions
obtained from processing of starch-based biomass wherein the
processed starch-based biomass is at least about 1% starch in
composition by weight. Examples of starch-based biomass are grains
and starchy roots of plants. Grains include but are not limited to
corn, wheat, barley, sorghum, and rice. Starchy roots are starch
storage root biomass such as potato, sweet potato, yam, taro,
carrot, turnip, and cassava. Examples of compositions obtained from
processing of starch-based biomass may include grain fractions and
by-products such as fiber fractions, endosperm fractions, wet cake,
distillers dry grains (DDG) or distillers dry grains with solubles
(DDGS)), and biomass post any liquefaction, saccharification,
fermentation, SSF process and/or other process or treatment of
starch-based biomass.
[0079] SSF process and/or other process or treatment of
starch-based biomass.
[0080] In various embodiments the starch-based biomass may be size
reduced, such as by hammer milling, shredding, chopping, chipping,
disc refining, and/or cutting. Particles of starch-based biomass
used may be from very small, in the micron range (i.e. 1-999
microns), to much larger, with at least one dimension on the order
of centimeters. In various embodiments the starch-based biomass may
be dried, such as air dried or dried with heat and optionally with
vacuum. The moisture content of starch-based biomass used in the
present process is generally less than 30%. In various embodiments
the moisture content of starch-based biomass used in the present
process is less than at least about 30%, 25%, 20%, 15%, 10%, or
less. Typical moisture content of dried starch-based biomass is
less than 15%. The moisture content is low enough to provide a
friablilty to the biomass. Techniques such as freezing may be
employed to increase the brittleness/friability of the starch-based
biomass to allow for milling of high moisture content biomass.
[0081] The mechanical forces applied to the starch-based biomass
are compression, and impact forces that result in normal and shear
stresses. Stress may be generated by rolling action against a
surface wherein forces are applied by rolling a mass over material
against a surface, where sandwiching the material between the mass
and the surface creates an anisotropic stress distribution (i.e.
shearing, sudden compression (impact) and decompression. The mass
is generally referred to as a media. Types of media that may be
used to apply compression and impact forces include, but are not
limited to, a puck, a ring, a roller, a rod, a disc, and a
sphere.
[0082] The particles that are ground via these mechanisms may also
shear against one another for additional destructuring. In one
embodiment at least one force applied is of at least about 5,000 N,
with stresses of at least 5,000 psi. There may be one level of
force applied, or more than one level of force which may be applied
concurrently. For example, there may be multiple media that apply
forces of different strengths, as described below and shown in FIG.
1. In the case of multiple forces, included is at least one force
of at least about 1,500 N with stresses of at least 5,000 psi.
There may be additional lower forces (as well as stresses) applied
concurrently. In one embodiment, whether a single level or multiple
levels of forces are applied, the forces include a level of at
least about 7,000 N combined with stresses of at least 5,000 psi.
In other embodiments with single or multiple forces, the forces
include a level of at least about 7,000, 10,000, 12,000, 15,000,
20,000, 25,000, 50,000, 100,000 N or greater combined with stresses
of at least about 5,000, 8,000, 13,000, 15,000, 18,000, 20,000,
22,000, or 25,000 psi. In the present process these forces are
imparted via a non-vibratory apparatus which does not contain any
free flowing media. Media of an apparatus used in the present
process are attached to a support, and the described forces are
generated using centrifugal, hydraulic, or spring mechanisms and
applied to the media and/or a grinding surface.
[0083] For illustrative purposes, a diagram of a small scale
apparatus for imparting compression, impact and shear forces using
centrifugal motion is shown in FIG. 1. This apparatus is a
vibratory mill which has free flowing media. A top view of the
apparatus is shown (10). A puck (12) is centrally located in a
chamber (18), which is attached to an eccentric motor. Two rings
(14 and 16) surround the puck, one inside the other, in the chamber
(18). When driven by the eccentric motor, the puck and rings
produce centrifugal motion, applying compression and impact forces
to biomass contained within the chamber.
[0084] An example of a non-vibratory apparatus without free flowing
media for imparting compression, impact and shear forces that may
be used in the present process is a ring-roller mill (Perry's
Chemical Engineers' Handbook, 8.sup.th Edition, p 21-60). One mill
of this type which generates centrifugal force is a centrifugal
force roller mill.
[0085] In one embodiment, force is applied through centrifugal
motion in a centrifugal force roller mill using at least one
grinding medium that is attached to a rotating shaft. The grinding
medium revolves around the rotating shaft with an angular frequency
to create a centrifugal force and swings against a grinding surface
thereby applying compression, impact, and shearing forces to
starch-based biomass which is between the medium and grinding
surface in the apparatus. In one embodiment the medium is an object
with a mass of at least 100 kilograms.
[0086] An example of this apparatus is shown in FIG. 2, with a side
view in A and a top view in B (the two arrows labeled 2B in A show
the region of the top view). A drive shaft (20) that rotates (32)
is attached to a cross bar (22) that has a hinge (26) at each end.
The hinge at each end attaches a rod (24) to the cross bar.
Attached to the rod is a roller (28) which rotates (34). As the
shaft turns, the rod swings (36) at the hinge and the roller
contacts the grinding surface (30). In FIG. 2B is shown that the
grinding surface (30) is a continuous ring that the rollers
continuously make contact with as the drive shaft (20) rotates. The
shape of the roller can be tapered in any manner and the grinding
surface can be angled in any manner such that when the drive shaft
rotates and the rollers swing out, the roller makes contact with
the grinding surface. Contact may be either over the total surface
of the roller, or over a substantial portion of the surface of the
roller. Preferably, the contact is substantially uniform along the
length of the roller.
[0087] The centrifugal force of this type of mill is related to the
mass of the medium, the center-of-mass radius of the medium, and
the angular frequency of rotation, as described in Equation 1, and
will vary depending on these aspects of an apparatus used in the
present process. The magnitude of centrifugal force [N] is
described by:
F=mr.omega..sup.2 [Equation 1]
where m represents the mass [kg] of the media, r represents the
center-of-mass radius of the media [m], and .omega. represents the
angular frequency [rad/s] of rotation.
[0088] From the Hertzian contact equations, the contact stress
formed by contact of two cylinders is calculable. For a concave
cylinder (ring) and a convex cylinder (roller) the calculation of
the Hertzian contact half-width, b, and stress, .sigma..sub.H, is
calculated by the following equations:
b = 2 F .pi. i .times. ( 1 - v 1 2 ) E 1 + ( 1 - v 2 2 ) E 2 1 d 1
- 1 d 2 Equation 2 .sigma. H = 2 F .pi. i .times. 1 d 1 1 d 2 ( 1 -
v 1 2 ) E 1 + ( 1 - v 2 2 ) E 2 Equation 3 ##EQU00001##
[0089] Where F is the force between the two cylinders (centrifugal
force in this case), E.sub.1 and E.sub.2 are the elastic moduli for
the roller and ring, respectively, d.sub.1 and d.sub.2 are the
diameters of the convex and concave surfaces (e.g. roller and ring
(or chamber)), respectively, l is the contact length along the
cylinders, and v.sub.1 and v.sub.2 are the Poisson ratios for the
roller and ring, respectively.
[0090] In an apparatus with low masses of rollers and low center of
mass radii, the G-forces required to achieve a desired force are
high, (for example, greater than 15 G) such as a combination of
.about.1,500 N and G-forces of 21.5 G. However lower G-force is
desired to reduce the power requirement. In one embodiment an
apparatus applies to the starch-based biomass greater than 5,000 N
and the G-force is less than 10 G. In another embodiment an
apparatus applies to the starch-based biomass at least about 10,000
N and the G-force is about 5 G.
[0091] A centrifugal force roller mill can contain a wide variety
of dimensions based on the scale of the operation, such as a ring
diameter of between about 12 and120 inches, roller diameters of
between about 4 and 40 inches, and heights of the ring and roller
of between about 3 and 30 inches. Mills with different dimensions
can be operated with rpm values from about 50 to about 500 rpm. A
mill used in the present process additionally may be equipped with
a gas conveying system with a recycle classifier and cyclone for
product recovery. The gas composition and feed rate may be adjusted
to prevent dust explosions. One skilled in the art can choose the
mill size, rpm, and required motors to drive the mill to obtain the
desired force, which is governed by Equation 1 above. The residence
time in the mill can be tuned by modifying classifier and air
handling systems, to ensure that the starch-based material that
exits the mill contains destructured starch. One of skill in the
art can modify the system to achieve the required force, residence
time, and power input to achieve energy input of less than 40% of
the starting total combustible energy of the input starch-based
biomass.
[0092] To achieve processed energy equivalency greater than 60% for
starch-based biomass, mill dimensions are tailored to reach high
performance in a centrifugal force roller mill. The ratio between
the radius of the media and radius of the ring or chamber, herein
referred to as Radii Ratio, is a defining parameter in stress
calculations (Equation 4).
RadiiRatio=radius.sub.media/radius.sub.chamber Equation 4
[0093] When operating the roller mill with high centrifugal force,
a preferred geometric arrangement for any force magnitude is when
the Radii Ratio equals 1:3 (0.33). At a Radii Ratio of 0.33 the
pressure reaches a maximum. For a non-vibratory mill with a central
driving shaft, the maximum Radii Ratio is .about.1:2 (0.5), defined
by the distance between the chamber radius and the central shaft.
Thus in the present process wherein centrifugal motion is used to
impart the described forces, the Radii Ratio is less than 0.5. In
one embodiment the Radii Ratio is less than 0.4. In one embodiment
the Radii Ratio is about 0.33, which is used to achieve maximal
pressure.
[0094] Mill dimensions that approximately represent a Radii Ratio
of 1:3 are desired to achieve an energy equivalency greater than
60% for processed starch-based biomass. In one embodiment, the mill
cfRRM-1 in Table 1 represents a mill with a chamber diameter of 40
inches, roller height of 10 inches, and operates at 90 rpm. For
this fixed rpm, roller height, and chamber diameter, the optimal
roller radius to achieve a peak pressure would be 6.67 inches
(16.94 cm) (Radii Ratio=1/3 or 0.33). The minimum Radii Ratio for
the cfRRM-1's fixed ring diameter, roller height, and rpm would be
0.3129 to achieve a minimum of 5,000 N and 5,000 psi (34.47 MPa).
The centrifugal force on the system can be changed by increasing or
decreasing the contact length of the mass-ring interface (i.e.
making the roller and grinding surface longer). For example,
increasing the contact length effectively increases the mass of the
roller and hence increases the centrifugal force (assuming all else
is constant, see Equation 1). Changing of the contact length while
all else is kept constant will not affect the Hertzian stress
calculation.
[0095] In another embodiment, the mill cfRRM-4 in Table 1
represents a mill with a chamber diameter of 100 inches, roller
height of 29.2 inches, and operates at 100 rpm. For the fixed rpm,
roller height, and chamber diameter, the optimal roller radius to
achieve a peak pressure would be 16.67 inches (42.34 cm) (Radii
Ratio=1/3 or 0.33). The minimum Radii Ratio for the cfRRM-4's fixed
ring diameter, roller height, and rpm would be 0.0610 to achieve a
minimum of 5,000 N and 5,000 psi (34.47 MPa).
[0096] With an optimized Radii Ratio, such as those in Table 1,
much higher forces and stresses can be achieved. In various
embodiments the stress is greater than about 5,000, 8,000, 13,000,
15,000, 18,000, 20,000, 22,000, or 25,000 psi (34.37, 55.16, 89.63,
103.42, 124.11, 137.90, 151.68, 172.37 MPa).
[0097] Equation 6 shows a modified Hertzian equation from Equation
3, which illustrates the embodiments mentioned above. Equation 6 is
derived from combining Equations 4 and 5 with Equation 3, to yield
a simplified equation relating Hertzian contact theory to the Radii
Ratio that defines a mill.
F = .pi. * ( radius mass ) 2 * l * ( radius chamber - radius mass )
* .omega. 2 Equation 2 Pressure = radius chamber * .omega. * .rho.
* ratio * ( ratio - 1 ) 2 ( 1 - v 1 2 ) E 1 + ( 1 - v 2 2 ) E 2
Equation 6 ##EQU00002##
[0098] In another embodiment, the force is applied using spring
and/or hydraulic mechanisms. An apparatus for applying force in
this manner typically has one or more milling media (e.g. rollers)
that are forced against a grinding surface (e.g. a ring). The
apparatus may function by moving the grinding surface (such as by
rotating a shaft to which the ring is attached) while the grinding
media remain motionless, which causes material to be fed through
and ground between the media and the surface. An apparatus of this
type is available commercially such as from FLSmidth (Bethlehem,
Pa.), ALSTOM Power Inc. (Windsor, Conn.), and Babcock & Wilcox
(Charlotte, N.C.). Alternatively, this type of apparatus may
function by moving grinding media attached to a central rotating
shaft and keeping the grinding ring stationary. Further, both the
media and the surface may move, but motion relative to each other
is required to cause grinding.
[0099] In one embodiment each individual application of force
occurs in less than ten milliseconds. More typically, each
individual application of force occurs in between 1 and 200
microseconds. In one embodiment the compression, impact, and
shearing forces are applied with a specific energy input that is
less than 40% of the total combustible energy of the starch-based
biomass being treated, as further described below. A low energy
input is achieved by application of the described high forces
together with a throughput of starch-based biomass that achieves a
power draw that is less than 40% of the total combustible energy of
the starch-based biomass being treated. The high forces (>5,000
N) are used to achieve a stress of at least 5,000 psi. This process
performs mechanical destructuring of the starch-based biomass such
that it produces a destructured biomass product. At constant power,
for lower time periods over which forces are applied, the specific
energy requirement is lowered compared to methods requiring longer
times, thereby providing a more cost-effective, energy-efficient
pretreatment process.
[0100] In one embodiment the compression and impact forces applied
to the starch-based biomass are applied by surfaces which are not
intentionally textured as in a cog ring or gear type media. Thus
the surfaces of the media and the grinding surface are considered
to be smooth, although there may be imperfections in these
surfaces. Owing to the isotropic form of a smooth surface of a
circular ring, the wear resistance is expected to be higher.
[0101] The specific energy input [kJ/g of dry biomass] required to
convert an amount of starch-based biomass to a destructured product
may be calculated by dividing the steady state power output [kW] of
the apparatus used by the throughput [g/s]. The power output for
any auxiliary equipment required for the operation of the
apparatus, such as a blower and classifier, is added to the power
output of the mill. The specific energy input is reduced for a high
force centrifugal apparatus as compared to other processes.
[0102] In the present process the destructured starch-based biomass
is produced with high compression and impact forces in combination
with some shear from the rolling action of the media against a
surface. The mechanical energy input that is required to achieve
starch-based biomass destructuring can be compared to the total
combustible energy of the biomass. The total combustible energy of
the biomass, or higher heating value (HHV) or Gross Calorific Value
(GCV), can be measured according to the American Society for
Testing and Materials procedure ASTM-D2015. For example, the total
combustible energy value for corn is generally 18,000 to 20,000
kj/kg. The present process is performed such that the energy input
for obtaining destructured corn is less than 40% of this value,
such that the destructured starch-based biomass has a processed
energy equivalency of greater than 60%. Energy input may be less
than 40%, 35%, 30%, 25% or less, which gives 60%, 65%, 70%, 75% or
greater energy equivalency.
[0103] In one embodiment energy input of less than 40% of total
combustible energy of the biomass feed is used to produce a
destructured biomass in a ring and puck mill, wherein a force of at
least 1,500N creates a stress of greater than 5,000 psi. The force
may be at least 1,500, 3,000, 5,000, 10,000, 12,000, 15,000,
20,000, 25,000, 50,000, 100,000 N or greater. The stress may be
greater than 5,000, 8,000, 13,000, 15,000, 18,000, 20,000, 22,000,
or 25,000 psi. Energy input may be less than 40%, 35%, 30%, 25% or
less which gives 60%, 65%, 70%, 75% or greater energy equivalency,
respectively.
[0104] Starch-based biomass treated using the present process
contains destructured starch. The destructured starch-based biomass
has crystallinity that is reduced, as compared to the crystallinity
of the starch-based biomass which is not treated, by at least about
25%. In various embodiments, crystallinity of starch-based biomass
may be reduced by at least about 25%, 30%, 35%, 40%, 45%, 50%, or
greater.
[0105] Starch-based biomass may contain a fiber component that
contains any of cellulose, hemicellulose, and lignin. This
component is cellulosic biomass, and occurs for example in the
embryo or germ of a grain. The forces used in the present process
also act to destructure cellulosic (including lignocellulosic)
biomass that is present in a starch-based biomass. In one
embodiment is a destructured starch-based biomass that includes
destructured starch and a destructured cellulosic biomass
component. Sugars may be released from the destructured cellulosic
biomass component using saccharification enzymes that degrade
cellulose and/or hemicellulose. Enzymes that may be used include
saccharification enzyme preparations that are commercially
available such as Spezyme.RTM. CP cellulase, Multifect.RTM.
xylanase, Accelerase.RTM. 1500, Accellerase.RTM. DUET, and
Accellerase.RTM. Trio.TM. (Dupont.TM./Genencor.RTM., Wilmington,
Del.), and Novozyme-188 (Novozymes, 2880 Bagsvaerd, Denmark). In
addition, the saccharification enzymes may be unpurified and
provided as a cell extract or a whole cell preparation. The enzymes
may be engineered and may be produced using recombinant
microorganisms that have been engineered to express one or more
saccharifying enzymes.
[0106] Starch-based biomass may contain an oil component, such as
in the embryo or germ of a grain. In destructured starch-based
biomass more oil is substantially free, as compared to oil in
starch-based biomass that is not destructured as described herein.
Free oil is defined as oil that is obtained by a gentle hexane
extraction, such as oil obtained by following the method of "Oil
analysis" as described in General Methods herein.
Animal Feed or Feed Additive
[0107] In some embodiments the destructured starch-based biomass
obtained in accordance with the present invention and/or the
starch-based biomass hydrolysate (which refers to a product
resulting from saccharification (suitably enzyme saccharification)
of the destructured starch-based biomass) is used in feed (e.g.
animal feed) applications, e.g. as a feed additive composition, a
premix, or a feedstuff or a part of any one of a feed additive
composition, a premix, or a feedstuff.
[0108] The destructed starch-based biomass and/or the starch-based
biomass hydrolysate may be used in combination with at least one
in-feed enzyme.
[0109] When the destructured starch-based biomass or the
starch-based biomass hydrolysate is used in combination with the at
least one in-feed enzyme, the combination may be fed to an animal
immediately after admixing the destructured starch-based biomass or
the starch-based biomass hydrolysate with the at least one in-feed
enzyme.
[0110] In other embodiments the in-feed enzyme is maintained in an
inactive form on the destructured starch-based biomass or the
starch-based biomass hydrolysate, until such time that the enzyme
enters the gastrointestinal tract (GIT) of an animal. In such
instances, when the enzyme enters the GIT of the animal the enzyme
is activated (e.g. becomes active).
[0111] In other embodiments, the in-feed enzyme is selected from
the group consisting of .alpha.-amylases (E.G. 3.2.1.1),
.beta.-amylases (E.G. 3.2.1.2), .gamma.-amylases (E.G. 3.2.1.3),
glucoamylases, cellulases, in particular endo-1,4-.beta.-glucanases
(EC 3.2.1.4) or endo-1,3-.beta.-glucanases (3.2.1.6), xylanases, in
particular endo-1,4-.beta.-glucanases (EC 3.2.1.8) or
xylan-endo-1,3-.beta.-xylosidases (EC 3.2.1.32), hemicellulases,
.alpha.-galactosidases (EC 3.2.1.22), polygalacturonases (EC
3.2.1.15), cellulose-1,4-.beta.-cellobiosidase (EC 3.2.1.91),
endoglucanases, in particular endo-1,6-6-glucanases (EC 3.2.1.75),
endo-1,2-.beta.-glucanases (EC 3.2.1.71),
endo-1,3-.beta.-glucanases (EC 3.2.1.39) or
endo-1,3-.alpha.-glucanases (EC 3.2.1.59), lipases, proteases, in
particular subtilisins (E.G. 3.4.21.62), bacillolysins (E.G.
3.4.24.28), alkaline serine proteases (E.G. 3.4.21.x), keratinases
(E.G. 3.4.x.x) or metalloproteases, mannanases, in particular
endo-beta-mannanases, esterases, exo-mannanases, galactanases,
xylanases, transferases, alpha-galactosidases, arabinosidases, aryl
esterases, beta-galactosidases, catalases, cellobiohydrolases,
cutinases, keratinases, laccases, lactases, ligninases,
lipoxygenases, oxidases, pectate lyases, pectin acetyl esterases,
pectinases, pentosanases, peroxidases, phenoloxidases,
phosphatases, phospholipases, polygalacturonases, pullulanases,
rhamnogalacturonases, tannases, transglutaminases, xylan
acetyl-esterases, xyloglucanases, xylosidases, additional phytases,
and combinations thereof.
[0112] In a preferred embodiment, the in-feed enzyme is a
glucoamylase.
[0113] The term "in-feed" as used herein means that the enzyme is
functional, preferably primarily functional, more preferably solely
functional, in the GIT of the animal. In other words, the term
"in-feed" as used herein means that the enzyme is substantially
inactive (or is inactive) in the feed additive composition and/or
on the destructured starch-based biomass or the starch-based
biomass hydrolysate prior to feeding the feed additive composition
or feedstuff comprising same to an animal.
[0114] The term "primarily functional" means that the enzyme mainly
functions on its substrate once it enters the GIT. In other words,
prior to entering the GIT the level of enzyme activity defined as
the amount of solubilisation of biomass material to
oligosaccharides and monosaccharides is less than 20%, suitably
less than 10%, preferably less than 5%, of the level of enzyme
activity after it enters the GIT (particularly, after it enters the
small intestine of the GIT).
[0115] The term "solely functional" as used herein means that the
enzyme is inactive before entering the GIT and is activated upon
entering the GIT.
[0116] The term "inactive" as used herein means that the enzyme is
not active. This may mean that the enzyme's activity is somehow
inhibited or that the enzyme is in an environment in which it is
inactive or that the enzyme is presented to its substrate
immediately prior to feeding to the animal such that there is not
enough time to be active. The "inactivity" of the enzyme is in any
event reversible once it enters the GIT of an animal.
[0117] The term "substantially inactive" as used herein means that
the enzyme has low activity compared with its activity once it has
entered the GIT (e.g. in the small intestine of the animal). For
instance, substantially inactive may mean that the enzyme in the
feed additive composition and/or on the lignocellulosic biomass has
less than 10% of its activity when compared with its activity in
the GIT (particularly, in the small intestine of the GIT).
[0118] Maintaining the "in-feed" enzyme in an inactive or
substantially inactive state in the feed additive composition
and/or on the destructured starch-based biomass or the starch-based
biomass hydrolysate can be achieved in a number of ways known to
one skilled in the art.
[0119] By way of example only maintaining the water content (wt %)
of the destructured starch-based biomass or the starch-based
biomass hydrolysate and/or of the in-feed enzyme and/or of the feed
additive composition at less than 15%, preferably less than 10%, is
sufficient to ensure that the in-feed enzyme is inactive or
substantially inactive in the feed additive composition and/or on
the destructured starch-based biomass or on the starch-based
biomass hydrolysate.
[0120] Therefore in one embodiment the in-feed enzyme may be
admixed with the destructured starch-based biomass or the
starch-based biomass hydrolysate when the destructured starch-based
biomass or the starch-based biomass hydrolysate, the in-feed enzyme
or both are in a dry state or a substantially dry state.
[0121] In one embodiment the destructured starch-based biomass or
the starch-based biomass hydrolysate and/or the in-feed enzyme
and/or the feed additive composition, post-admixing the
destructured starch-based biomass or the starch-based biomass
hydrolysate and the in-feed enzyme, are (maintained and/or stored)
in a dry state or substantially dry state.
[0122] The term "dry state" as used herein means that the
destructured starch-based biomass or the starch-based biomass
hydrolysate and/or the in-feed enzyme and/or the feed additive
composition contains no or only a very low amount of water. In
other words the term "dry state" as used herein may mean that the
destructured starch-based biomass or the starch-based biomass
hydrolysate and/or the in-feed enzyme and/or the feed additive
composition comprises less than 5%, preferably less than 1%, water
content (wt %).
[0123] The term "substantially dry state" as used herein means that
the destructured starch-based biomass or the starch-based biomass
hydrolysate and/or the in-feed enzyme and/or the feed additive
composition contains only a very low amount of water. In other
words the term "substantially dry state" as used herein may mean
that the destructured starch-based biomass or the starch-based
biomass hydrolysate and/or the in-feed enzyme and/or the feed
additive composition comprises less than 15%, preferably less than
10%, water content (wt %).
[0124] In one embodiment, the method according to the present
invention may comprise drying the destructured starch-based biomass
or the starch-based biomass hydrolysate prior to, during or after
(preferably prior to) admixing the biomass with at least one
in-feed enzyme.
[0125] In another embodiment the destructured starch-based biomass
or the starch-based biomass hydrolysate either before or after
adding the at least one in-feed enzyme comprises less than 15 wt %
moisture content.
[0126] In another embodiment the destructured starch-based biomass
or the starch-based biomass hydrolysate either before or after
adding the at least one in-feed enzyme comprises less than 10 wt %
moisture content.
[0127] In another embodiment the destructured starch-based biomass
or the starch-based biomass hydrolysate either before or after
adding the at least one in-feed enzyme comprises less than 5 wt %
moisture content.
[0128] In another embodiment the destructured starch-based biomass
or the starch-based biomass hydrolysate either before or after
adding the at least one in-feed enzyme comprises less than 1 wt %
moisture content.
[0129] The "in-feed" enzyme may be maintained in an inactive or
substantially inactive state in the feed additive composition
and/or on the destructured starch-based biomass or the starch-based
biomass hydrolysate by physically preventing the enzyme from
interacting with its substrate. For example the in-feed enzyme may
be encapsulated prior to admixing with the destructured
starch-based biomass or the starch-based biomass hydrolysate.
[0130] When the in-feed enzyme is physically prevented from
interacting with its substrate in the destructured starch-based
biomass or the starch-based biomass hydrolysate, then once in the
GIT the physical barrier is removed thus allowing the interaction
of the in-feed enzyme with its substrate.
[0131] By way of example only, the encapsulation may be removed by
passage of the encapsulated enzyme through the stomach of an
animal. The stomach of an animal is at very low (acidic) pH (e.g.
pH 2-4). This acidity can be used to activate encapsulated
enzymes.
[0132] In one embodiment the enzyme may be encapsulated by a
polymer, such as chitin or chitosans, gelatin, gum arabic or wax
for example. By way of example only the polymer may be a gelatin or
gum arabic as taught in Xue et al Food Funct. 2013, Apr. 25; 6
February (epub); 4 (4) 610-7 (which is incorporated herein by
reference). Alternatively, the polymer may a chitosan-based
hydrogel as taught in Zhang et al Biomacromolecules 2011,
12,2894-2901 (which is incorporated herein by reference).
[0133] In one embodiment the at least one in-feed enzyme may be
activated by feeding the at least one in-feed enzyme to an
animal.
[0134] The term "inactive" as used herein may mean that the enzyme
is presented to its substrate immediately prior to feeding to the
animal such that there is not enough time to be active before it
enters the GIT of the animal.
[0135] In one embodiment the at least one in-feed enzyme may be
admixed with the destructured starch-based biomass or the
starch-based biomass hydrolysate immediately prior to feeding the
feed additive composition or feedstuff comprising the destructured
starch-based biomass or the starch-based biomass hydrolysate to an
animal.
[0136] In one preferred embodiment the in-feed enzyme is maintained
in an inactive or substantially inactive state by maintaining the
feed additive composition in a dry state or substantially dry
state. This has additional benefits in that the handling, e.g.
processing, packaging, storage and transport of a dry composition
is easier than non-dry formulations (e.g. liquids).
[0137] However, it can also be envisaged that a kit where the
destructured starch-based biomass or the starch-based biomass
hydrolysate is physically separated from the in-feed enzyme (e.g.
by being in separate containers) would enable the end user (e.g.
farmer) to admix the destructured starch-based biomass or the
starch-based biomass hydrolysate with the in-feed enzyme
immediately prior to feeding the mixture to the animal. In such a
situation the destructured starch-based biomass or the starch-based
biomass hydrolysate and/or the in-feed enzyme may be in any
formulation, e.g. solid, semi-solid or liquid for example.
[0138] The destructured starch-based biomass or the starch-based
biomass hydrolysate can have even more nutritional value for use as
a feed if one combines this biomass with in-feed enzymes.
In-Feed Enzymes
[0139] In one embodiment the enzyme composition comprises at least
two (suitably at least three) enzymes. The enzyme composition
comprising the at least two (suitably at least three) enzymes
preferably has at least two (suitably at least three) of the
following: an endoglucanase, an endoxylanase or a
.beta.-glucosidase.
[0140] In one embodiment the at least one in-feed enzyme(s) may be
a single enzyme or a combination of enzymes (e.g. an enzyme
mix).
[0141] In one preferred embodiment the at least one in-feed enzyme
is an enzyme mixture.
[0142] Preferably the in-feed enzyme in accordance with the present
invention is stable and active in the gastrointestinal tract (GIT)
of an animal. In one embodiment the in-feed enzyme is resistant to
pepsin. In one embodiment the in-feed enzyme is tolerant to bile
salts. In one embodiment the in-feed enzyme is resistant to low pH.
In one embodiment the in-feed enzyme can withstand pelleting
temperatures (70-95.degree. C.). In one embodiment the in-feed
enzyme is active in the range of 37-40.degree. C.
Biophysical Characteristics
[0143] The present invention also relates to uses and methods for
improving the biophysical characteristics of an animal by
administering to an animal an effective amount of a feed additive
composition or a feedstuff according to the present invention.
[0144] As used herein the term "biophysical characteristics" as
used herein means one or more of the group selected from the
following: performance of an animal, growth performance of an
animal, feed conversion ratio (FCR), ability to digest a raw
material (e.g. nutrient digestibility, including starch , fat,
protein, fibre digestibility), nitrogen retention, carcass yield,
growth rate, weight gain, body weight, mass, feed efficiency, body
fat percentage, body fat distribution, growth, egg size, egg
weight, egg mass, egg laying rate and environmental impact, e.g.
manure output and/or nitrogen excretion.
[0145] In one embodiment the biophysical characteristic of the
animal means the performance of the animal.
Performance
[0146] As used herein, "performance of the animal" may be
determined by the feed efficiency and/or weight gain of the animal
and/or by the feed conversion ratio and/or by the digestibility of
a nutrient in a feed (e.g. amino acid digestibility) and/or
digestible energy or metabolizable energy in a feed and/or by
nitrogen retention.
[0147] Preferably "performance of the animal" is determined by feed
efficiency and/or weight gain of the animal and/or by the feed
conversion ratio (FCR).
[0148] By "improved performance of the animal" it is meant that
there is increased feed efficiency, and/or increased weight gain
and/or reduced feed conversion ratio and/or improved digestibility
of nutrients or energy in a feed and/or by improved nitrogen
retention in the animal resulting from the use of feed additive
composition of the present invention compared with feeding the
animal the lignocellulosic biomass which has not been treated in
accordance with the present invention.
[0149] Preferably, by "improved animal performance" it is meant
that there is increased feed efficiency and/or increased weight
gain and/or reduced feed conversion ratio.
[0150] As used herein, the term "feed efficiency" refers to the
amount of weight gain in an animal that occurs when the animal is
fed ad-libitum or a specified amount of food during a period of
time.
[0151] By "increased feed efficiency" it is meant that the use of a
feed additive composition according the present invention in feed
results in an increased weight gain per unit of feed intake
compared with an animal fed with the lignocellulosic biomass which
has not been treated in accordance with the present invention.
Feed Conversion Ration (FCR)
[0152] As used herein, the term "feed conversion ratio" refers to
the amount of feed fed to an animal to increase the weight of the
animal by a specified amount.
[0153] An improved feed conversion ratio means a lower feed
conversion ratio.
[0154] By "lower feed conversion ratio" or "improved feed
conversion ratio" it is meant that the use of a feed additive
composition in feed results in a lower amount of feed being
required to be fed to an animal to increase the weight of the
animal by a specified amount compared to the amount of feed
required to increase the weight of the animal by the same amount
when the lignocellulosic biomass which has not been treated in
accordance with the present invention is used in or as the
feed.
Nutrient Digestibility
[0155] Nutrient digestibility as used herein means the fraction of
a nutrient that disappears from the gastro-intestinal tract or a
specified segment of the gastro-intestinal tract, e.g. the small
intestine. Nutrient digestibility may be measured as the difference
between what is administered to the animal and what comes out in
the faeces of the animal, or between what is administered to the
animal and what remains in the digesta on a specified segment of
the gastro intestinal tract, e.g. the ileum.
[0156] Nutrient digestibility as used herein may be measured by the
difference between the intake of a nutrient and the excreted
nutrient by means of the total collection of excreta during a
period of time; or with the use of an inert marker that is not
absorbed by the animal, and allows the researcher calculating the
amount of nutrient that disappeared in the entire gastro-intestinal
tract or a segment of the gastro-intestinal tract. Such an inert
marker may be titanium dioxide, chromic oxide or acid insoluble
ash. Digestibility may be expressed as a percentage of the nutrient
in the feed, or as mass units of digestible nutrient per mass units
of nutrient in the feed.
[0157] Nutrient digestibility as used herein encompasses starch
digestibility, fat digestibility, protein digestibility, fiber
digestibility and amino acid digestibility.
[0158] Energy digestibility as used herein means the gross energy
of the feed consumed minus the gross energy of the faeces or the
gross energy of the feed consumed minus the gross energy of the
remaining digesta on a specified segment of the gastro-intestinal
tract of the animal, e.g. the ileum. Metabolizable energy as used
herein refers to apparent metabolizable energy and means the gross
energy of the feed consumed minus the gross energy contained in the
faeces, urine, and gaseous products of digestion. Energy
digestibility and metabolizable energy may be measured as the
difference between the intake of gross energy and the gross energy
excreted in the faeces or the digesta present in specified segment
of the gastro-intestinal tract using the same methods to measure
the digestibility of nutrients, with appropriate corrections for
nitrogen excretion to calculate metabolizable energy of feed.
Nitrogen Retention
[0159] Nitrogen retention as used herein means an animal's ability
to retain nitrogen from the diet as body mass. A negative nitrogen
balance occurs when the excretion of nitrogen exceeds the daily
intake and is often seen when the muscle is being lost. A positive
nitrogen balance is often associated with muscle growth,
particularly in growing animals.
[0160] Nitrogen retention may be measured as the difference between
the intake of nitrogen and the excreted nitrogen by means of the
total collection of excreta and urine during a period of time. It
is understood that excreted nitrogen includes undigested protein
from the feed, endogenous proteinaceous secretions, microbial
protein, and urinary nitrogen.
Carcass Yield and Meat Yield
[0161] The term carcass yield as used herein means the amount of
carcass as a proportion of the live body weight, after a commercial
or experimental process of slaughter. The term carcass means the
body of an animal that has been slaughtered for food, with the
head, entrails, part of the limbs, and feathers or skin removed.
The term meat yield as used herein means the amount of edible meat
as a proportion of the live body weight, or the amount of a
specified meat cut as a proportion of the live body weight.
Weight Gain
[0162] The present invention further provides a method of
increasing weight gain in an animal, e.g. poultry or swine,
comprising feeding said animal a feedstuff comprising a feed
additive composition according to the present invention.
[0163] An "increased weight gain" refers to an animal having
increased body weight on being fed feed comprising a feed additive
composition compared with an animal being fed a feed comprising or
consisting of lignocellulosic biomass which has not been treated in
accordance with the present invention.
Improving
[0164] The term "improving" as used herein means improved compared
with feeding animal the lignocellulosic biomass which has not been
treated in accordance with the present invention.
Admixing
[0165] In one embodiment "admixing" as used herein includes any
method for admixing, such as mixing, combining, spraying etc.).
Animal
[0166] The term "animal", as used herein, means an animal that is
to be or has been administered with a feed additive composition
according to the present invention or a feedstuff comprising said
feed additive composition according to the present invention.
[0167] Preferably, the animal is a mammal (e.g. a non-human
mammal), bird, fish or crustacean including for example livestock
or a domesticated animal (e.g. a pet).
[0168] In one embodiment the "animal" is livestock.
[0169] The term "livestock", as used herein refers to any farmed
animal. Preferably, livestock is one or more of cows or bulls
(including calves), pigs (including piglets, swine), poultry
(including broilers, layers, chickens and turkeys), birds, fish
(including freshwater fish, such as salmon, cod, trout and carp,
e.g. koi carp, and marine fish, such as sea bass), crustaceans
(such as shrimps, mussels and scallops), horses (including race
horses), sheep (including lambs).
[0170] In one embodiment the animal is poultry (including broilers,
layers, chickens and turkeys).
[0171] In another embodiment the "animal" is a domesticated animal
or pet or an animal maintained in a zoological environment.
[0172] The term "domesticated animal or pet or animal maintained in
a zoological environment" as used herein refers to any relevant
animal including canines (e.g. dogs), felines (e.g. cats), rodents
(e.g. guinea pigs, rats, mice), birds, fish (including freshwater
fish and marine fish), and horses.
[0173] In one embodiment the animal is a monogastric animal. In a
preferred embodiment the monogastric animal may be poultry or pig
(or a combination thereof).
[0174] In another embodiment the animal is a ruminant animal.
Packaging
[0175] In one embodiment the enzyme composition and/or feed
additive composition and/or premix and/or feed or feedstuff
according to the present invention is packaged.
[0176] In one preferred embodiment feed additive composition and/or
feed ingredient and/or premix and/or feed or feedstuff is packaged
in a bag, such as a paper bag.
[0177] In an alternative embodiment the enzyme composition and/or
feed additive composition and/or feed ingredient and/or premix
and/or feed or feedstuff may be sealed in a container. Any suitable
container may be used.
Feed
[0178] The destructured starch-based biomass or the starch-based
biomass hydrolysate (with or without in feed enzyme(s)) may be used
as or in the preparation of a feed additive composition or a
feed.
[0179] The feed additive composition of the present invention may
be used as--or in the preparation of--a feed.
[0180] The term "feed" is used synonymously herein with
"feedstuff".
[0181] The feed may be in the form of a solution or as a
solid--depending on the use and/or the mode of application and/or
the mode of administration.
[0182] When used as--or in the preparation of--a feed--such as
functional feed--the destructured starch-based biomass or the
starch-based biomass hydrolysate (with or without in feed
enzyme(s)) of the present invention may be used in conjunction with
one or more of: a nutritionally acceptable carrier, a nutritionally
acceptable diluent, a nutritionally acceptable excipient, a
nutritionally acceptable adjuvant, a nutritionally active
ingredient.
[0183] In a preferred embodiment the destructured starch-based
biomass or the starch-based biomass hydrolysate (with or without in
feed enzyme(s)) or the feed additive composition of the present
invention is admixed with a feed component to form a feedstuff.
[0184] The term "feed component" as used herein means all or part
of the feedstuff. Part of the feedstuff may mean one constituent of
the feedstuff or more than one constituent of the feedstuff, e.g. 2
or 3 or 4. In one embodiment the term "feed component" encompasses
a premix or premix constituents.
[0185] Preferably the feed may be a fodder, or a premix thereof, a
compound feed, or a premix thereof. In one embodiment the feed
additive composition according to the present invention may be
admixed with a compound feed, a compound feed component or to a
premix of a compound feed or to a fodder, a fodder component, or a
premix of a fodder.
[0186] The term fodder as used herein means any food which is
provided to an animal (rather than the animal having to forage for
it themselves). Fodder encompasses plants that have been cut. The
term fodder includes hay, straw, silage, compressed and pelleted
feeds, oils and mixed rations, and also sprouted grains and
legumes.
[0187] Fodder may be obtained from one or more of the plants
selected from: alfalfa (Lucerne), barley, birdsfoot trefoil,
brassicas, Chau moellier, kale, rapeseed (canola), rutabaga
(swede), turnip, clover, alsike clover, red clover, subterranean
clover, white clover, grass, false oat grass, fescue, Bermuda
grass, brome, heath grass, meadow grasses (from naturally mixed
grassland swards, orchard grass, rye grass, Timothy-grass, corn
(maize), millet, oats, sorghum, soybeans, trees (pollard tree
shoots for tree-hay), wheat, and legumes.
[0188] The term "compound feed" means a commercial feed in the form
of a meal, a pellet, nuts, cake or a crumble. Compound feeds may be
blended from various raw materials and additives. These blends are
formulated according to the specific requirements of the target
animal.
[0189] Compound feeds can be complete feeds that provide all the
daily required nutrients, concentrates that provide a part of the
ration (protein, energy) or supplements that only provide
additional micronutrients, such as minerals and vitamins.
[0190] The main ingredients used in compound feed are the feed
grains, which include corn, soybeans, sorghum, oats, and
barley.
[0191] Suitably a premix as referred to herein may be a composition
composed of microingredients such as vitamins, minerals, chemical
preservatives, antibiotics, fermentation products, and other
essential ingredients. Premixes are usually compositions suitable
for blending into commercial rations.
[0192] Any feedstuff of the present invention may comprise one or
more feed materials selected from the group comprising a) cereals,
such as small grains (e.g., wheat, barley, rye, oats and
combinations thereof) and/or large grains such as maize or sorghum;
b) by products from plants (e.g. cereals), such as wet-cake,
distillers dried grain (DDG), and distillers dried grain solubles
(DDGS), corn fibre, corn germ meal, corn bran, Hominy feed, corn
gluten feed, wheat shorts, wheat middlings or combinations thereof
(preferably by products of methods according to the present
invention); c) protein obtained from sources such as soya,
sunflower, peanut, lupin, peas, fava beans, cotton, canola, fish
meal, dried plasma protein, meat and bone meal, potato protein,
whey, copra, sesame; d) oils and fats obtained from vegetable and
animal sources; e) minerals and vitamins.
[0193] A feedstuff of the present invention may contain at least
30%, at least 40%, at least 50% or at least 60% by weight corn and
soybean meal or corn and full fat soy, or wheat meal or sunflower
meal.
[0194] In addition or in the alternative, a feedstuff of the
present invention may comprise at least one high fibre feed
material and/or at least one by-product of the at least one high
fibre feed material to provide a high fibre feedstuff. Examples of
high fibre feed materials include: wheat, barley, rye, oats, by
products from plants (e.g. cereals), such as wet-cake, distillers
dried grain (DDG), and distillers dried grain solubles (DDGS), corn
fibre, corn germ meal, corn bran, Hominy feed, corn gluten feed,
wheat shorts, wheat middlings or combinations thereof. Some protein
sources may also be regarded as high fibre: protein obtained from
sources such as sunflower, lupin, fava beans and cotton.
[0195] In the present invention the feed may be one or more of the
following: a compound feed and premix, including pellets, nuts or
(cattle) cake; a crop or crop residue: corn, soybeans, sorghum,
oats, barley, corn stover, copra, straw, chaff, sugar beet waste;
fish meal; freshly cut grass and other forage plants; meat and bone
meal; molasses; oil cake and press cake; oligosaccharides;
conserved forage plants: hay and silage; seaweed; seeds and grains,
either whole or prepared by crushing, milling etc.; sprouted grains
and legumes; yeast extract.
[0196] The term "feed" in the present invention also encompasses in
some embodiments pet food. A pet food is plant or animal material
intended for consumption by pets, such as dog food or cat food. Pet
food, such as dog and cat food, may be either in a dry form, such
as kibble for dogs, or wet canned form. Cat food may contain the
amino acid taurine.
[0197] The term "feed" in the present invention also encompasses in
some embodiments fish food. A fish food normally contains macro
nutrients, trace elements and vitamins necessary to keep captive
fish in good health. Fish food may be in the form of a flake,
pellet or tablet. Pelleted forms, some of which sink rapidly, are
often used for larger fish or bottom feeding species. Some fish
foods also contain additives, such as beta carotene or sex
hormones, to artificially enhance the colour of ornamental
fish.
[0198] The term "feed" in the present invention also encompasses in
some embodiment bird food. Bird food includes food that is used
both in birdfeeders and to feed pet birds. Typically bird food
comprises of a variety of seeds, but may also encompass suet (beef
or mutton fat).
[0199] As used herein the term "contacting" refers to the indirect
or direct application of the destructured starch-based biomass or
the starch-based biomass hydrolysate (with or without in feed
enzyme(s)) of the present invention to the product (e.g. the feed).
Examples of the application methods which may be used, include, but
are not limited to, treating the product in a material comprising
the feed additive composition, direct application by mixing the
feed additive composition with the product, spraying the feed
additive composition onto the product surface or dipping the
product into a preparation of the feed additive composition.
[0200] In one embodiment the destructured starch-based biomass or
the starch-based biomass hydrolysate (with or without in feed
enzyme(s)) or the feed additive composition of the present
invention is preferably admixed with the product (e.g. feedstuff).
Alternatively, the destructured starch-based biomass or the
starch-based biomass hydrolysate (with or without in feed
enzyme(s)) or the feed additive composition may be included in the
emulsion or raw ingredients of a feedstuff.
[0201] For some applications, it is important that the in feed
enzyme(s) is made available on or to the surface of a product to be
affected/treated. This allows the composition to impart one or more
of the following favourable characteristics: biophysical
characteristics, e.g. wherein the biophysical characteristic is
selected from the group consisting of one or more of the following:
performance of an animal, growth performance of an animal, feed
conversion ratio (FCR), ability to digest a raw material (e.g.
nutrient digestibility, including starch , fat, protein, fibre
digestibility), nitrogen retention, carcass yield, growth rate,
weight gain, body weight, mass, feed efficiency, body fat
percentage, body fat distribution, growth, egg size, egg weight,
egg mass, egg laying rate and environmental impact, e.g. manure
output and/or nitrogen excretion.
[0202] The feed additive compositions of the present invention may
be applied to intersperse, coat and/or impregnate a product (e.g.
feedstuff or raw ingredients of a feedstuff) with a controlled
amount of enzyme(s).
[0203] Preferably, the enzyme composition and/or feed additive
composition of the present invention will be thermally stable to
heat treatment up to about 70.degree. C.; up to about 85.degree.
C.; or up to about 95.degree. C. The heat treatment may be
performed for up to about 1 minute; up to about 5 minutes; up to
about 10 minutes; up to about 30 minutes; up to about 60 minutes.
The term thermally stable means that at least about 75% of the
enzyme components that were present/active in the additive before
heating to the specified temperature are still present/active after
it cools to room temperature. Preferably, at least about 80% of the
enzyme components that were present and active in the additive
before heating to the specified temperature are still present and
active after it cools to room temperature.
[0204] In a particularly preferred embodiment the in-feed enzymes
may be formulated into an enzyme composition.
[0205] The enzyme composition and/or feed additive composition may
be homogenized to produce a powder.
[0206] In an alternative preferred embodiment, the enzyme
composition and/or feed additive composition may be formulated to
granules as described in WO2007/044968 (referred to as TPT
granules) incorporated herein by reference.
[0207] In another preferred embodiment when the enzyme composition
and/or feed additive composition is formulated into granules the
granules comprise a hydrated barrier salt coated over the protein
core. The advantage of such salt coating is improved
thermo-tolerance, improved storage stability and protection against
other feed additives otherwise having adverse effect on the
enzyme.
[0208] Preferably, the salt used for the salt coating has a water
activity greater than 0.25 or constant humidity greater than 60% at
20.degree. C.
[0209] Preferably, the salt coating comprises a
Na.sub.2SO.sub.4.
[0210] The method of preparing a feed additive composition may also
comprise the further step of pelleting the powder. The powder may
be mixed with other components known in the art. The powder, or
mixture comprising the powder, may be forced through a die and the
resulting strands are cut into suitable pellets of variable
length.
[0211] Optionally, the pelleting step may include a steam
treatment, or conditioning stage, prior to formation of the
pellets. The mixture comprising the powder may be placed in a
conditioner, e.g. a mixer with steam injection. The mixture is
heated in the conditioner up to a specified temperature, such as
from 60-100.degree. C., typical temperatures would be 70.degree.
C., 80.degree. C., 85.degree. C., 90.degree. C. or 95.degree. C.
The residence time can be variable from seconds to minutes and even
hours. Such as 5 seconds, 10 seconds, 15 seconds, 30 seconds, 1
minutes 2 minutes., 5 minutes, 10 minutes, 15 minutes, 30 minutes
and 1 hour.
[0212] It will be understood that the feed additive composition of
the present invention is suitable for addition to any appropriate
feed material.
[0213] As used herein, the term feed material refers to the basic
feed material to be consumed by an animal. It will be further
understood that this may comprise, for example, at least one or
more unprocessed grains, and/or processed plant and/or animal
material such as soybean meal or bone meal.
[0214] As used herein, the term "feedstuff" refers to a feed
material to which one or more feed additive compositions or
destructured starch-based biomass or the starch-based biomass
hydrolysate (with or without in feed enzyme(s)) have been
added.
[0215] It will be understood by the skilled person that different
animals require different feedstuffs, and even the same animal may
require different feedstuffs, depending upon the purpose for which
the animal is reared.
[0216] Preferably, the feedstuff may comprise feed materials
comprising maize or corn, wheat, barley, triticale, rye, rice,
tapioca, sorghum, and/or any of the by-products, as well as protein
rich components like soybean mean, rape seed meal, canola meal,
cotton seed meal, sunflower seed mean, animal-by-product meals and
mixtures thereof. More preferably, the feedstuff may comprise
animal fats and/or vegetable oils.
[0217] Optionally, the feedstuff may also contain additional
minerals such as, for example, calcium and/or additional
vitamins.
[0218] Preferably, the feedstuff is a corn soybean meal mix.
[0219] In another aspect there is provided a method for producing a
feedstuff. Feedstuff is typically produced in feed mills in which
raw materials are first ground to a suitable particle size and then
mixed with appropriate additives. The feedstuff may then be
produced as a mash or pellets; the later typically involves a
method by which the temperature is raised to a target level and
then the feed is passed through a die to produce pellets of a
particular size. The pellets are allowed to cool. Subsequently
liquid additives such as fat and enzyme may be added. Production of
feedstuff may also involve an additional step that includes
extrusion or expansion prior to pelleting--in particular by
suitable techniques that may include at least the use of steam.
[0220] The feedstuff may be a feedstuff for a monogastric animal,
such as poultry (for example, broiler, layer, broiler breeders,
turkey, duck, geese, water fowl), swine (all age categories), a pet
(for example dogs, cats) or fish, preferably the feedstuff is for
poultry.
Formulation
[0221] In one embodiment the destructured starch-based biomass or
the starch-based biomass hydrolysate (with or without in feed
enzyme(s)) and/or feed additive composition and/or premix and/or
feedstuff in accordance with the present invention are dried.
[0222] The term "drying" means that the water content (wt %) of the
destructured starch-based biomass product (with or without in feed
enzyme(s)) or starch-based biomass hydrolysate product (with or
without in feed enzyme(s)) and/or feed additive composition and/or
premix and/or feedstuff in accordance with the present invention is
reduced to at less than 15%, preferably less than 10%.
[0223] Therefore the present invention yet further provides a dried
or substantially dried destructured starch-based biomass product
(with or without in feed enzyme(s)) or starch-based biomass
hydrolysate product (with or without in feed enzyme(s)) or feed
additive composition or premix or feedstuff.
[0224] The term substantially-dried in accordance with the present
invention means that the water content (wt %) of the product/feed
additve composition/premix/feedstuff is less than 30%, preferably
less than 15%, preferably less than 10%, preferably less than
5%.
[0225] In one embodiment the in-feed enzyme composition may be in a
dry enzyme formulation (e.g. in the form of granules or on a
carrier (such as a wheat carrier)) prior to admixing with the
destructured starch-based biomass or starch-based biomass
hydrolysate.
[0226] In one embodiment the saccharification enzyme composition
may be in a liquid enzyme formulation prior to admixing with the
destructured starch-based biomass.
[0227] In another embodiment the in-feed enzyme composition may be
in a liquid formulation prior to admixing with the destructured
starch-based biomass or starch-based biomass hydrolysate.
[0228] In another embodiment the saccharification enzyme
composition may be in a liquid formulation prior to admixing with
the destructured starch-based biomass.
[0229] In some embodiments the present invention may provide a
semi-liquid product or slurry product. A semi-liquid product or
slurry product in accordance with the present invention is a
product the water content (wt %) of which is at less than 90%,
preferably less than 80%, preferably less than 70% or more
preferably less than 60%.
[0230] When the enzyme is in a liquid formulation prior to admixing
with the the destructured starch-based biomass or starch-based
biomass hydrolysate, the enzyme may be admixed by spraying the
enzyme formulation or dipping the destructured starch-based biomass
or starch-based biomass hydrolysate into the enzyme formulation for
example.
[0231] The destructured starch-based biomass or starch-based
biomass hydrolysate (before or after enzyme treatment) and/or the
dried solid fraction may be milled and/or powdered and/or formed
into a meal.
[0232] In one embodiment the product and/or feed additive
composition and/or premix and/or feedstuff in accordance with the
present invention, are packaged and/or stored in a dry state or
substantially dry state.
[0233] The terms "dried" or "dry state" as used herein means that
the product and/or feed additive composition and/or premix and/or
feedstuff in accordance with the present invention contain no or
only a very low amount of water. In other words the term "dried" or
"dry state" as used herein may mean that the product and/or feed
additive composition and/or premix and/or feedstuff in accordance
with the present invention comprises less than 5%, preferably less
than 1%, water content (wt %).
[0234] The term "substantially dry state" as used herein means that
the product and/or feed additive composition and/or premix and/or
feedstuff in accordance with the present invention contains only a
very low amount of water. In other words the term "substantially
dry state" as used herein may mean that the and/or feed additive
composition and/or premix and/or feedstuff in accordance with the
present invention comprises less than 30%, preferably less than
15%, preferably less than 10%, water content (wt %).
[0235] In one embodiment the product and/or feed additive
composition and/or premix and/or feedstuff in accordance with the
present invention comprises less than 20 wt % moisture content.
[0236] In another embodiment the product on and/or feed additive
composition and/or premix and/or feedstuff in accordance with the
present invention comprises less than 15 wt % moisture content.
[0237] In another embodiment the product and/or feed additive
composition and/or premix and/or feedstuff in accordance with the
present invention comprises less than 10 wt % moisture content.
[0238] In another embodiment the product and/or feed additive
composition and/or premix and/or feedstuff in accordance with the
present invention comprises less than 5 wt % moisture content.
Sugar Production using High Force Milled Starch-Based Biomass
[0239] In some embodiments the destructured starch-based biomass
obtained in accordance with the present process is saccharified to
produce sugar-containing compositions. These compositions may be
called syrups. Different types of syrups may be produced with use
of enzymes having different hydrolytic activities. The exact
composition of the products of saccharification depends on the
combination of enzymes used, as well as the type of destructured
starch-based biomass processed. In various embodiments syrups are
produced by processes that omit at least one of: 1) liquefaction
and 2) additional alpha-amylase. Destructured starch-based biomass
produced using the present method provides a material that is a
better substrate for saccharification than a starch-based biomass
that is not destructured using the present process.
[0240] In one embodiment the syrup is a composition comprising
glucose (a glucose composition) produced by contacting destructured
starch-based biomass with glucoamylase and optionally at least one
of an alpha-amylase and a phytase. In various embodiments the
produced glucose composition has a DP1 concentration higher than
would result when saccharifying a whole ground corn liquefact
composition in place of the destructured starch-based biomass
composition; wherein the higher DP1 concentration is at least 0.1%,
0.2%, 0.4%, or 0.5% higher. In one embodiment the glucose
composition has a DP2 concentration lower than would result when
saccharifying a whole ground corn liquefact composition in place of
the destructured starch-based biomass; wherein DP2 concentration at
least 0.1% lower.
[0241] In some embodiments a glucose composition is referred to as
a composition comprising fermentable sugars. In addition to contact
with a glucoamylase, for production of a composition comprising
fermentable sugars, destructured starch-based biomass may be
contacted with at least one of an alpha-amylase and a phytase. The
glucose composition, or composition comprising fermentable sugars,
produced by methods described herein may be used in a fermentation
process to produce a product, called herein an End of Fermentation
(EOF) product. The glucose composition or composition comprising
fermentable sugars may also be called mash.
[0242] In one embodiment the syrup is a composition comprising
maltose (maltose composition) produced by contacting destructured
starch-based biomass with a maltogenic alpha-amylase. In various
embodiments the produced maltose composition has a DP2
concentration higher than would result when saccharifying a whole
ground corn liquefact composition in place of the destructured
starch-based biomass composition; wherein the higher DP2
concentration is at least 0.1% higher.
[0243] In one embodiment the syrup is a composition comprising
maltotriose (maltotriose composition) produced by contacting
destructured starch-based biomass with a beta-amylase and
optionally a pullulanase. In various embodiments the produced
maltotriose composition has lower DP1 and DP2 concentrations than
would result when saccharifying a whole ground corn liquefact
composition in place of the destructured starch-based biomass
composition; wherein the DP1 and DP2 concentrations are at least
0.1% lower.
[0244] In one embodiment the syrup is a composition comprising
maltotetraose (maltotetraose composition) produced by contacting
destructured starch-based biomass with a DP4 producing
alpha-amylase and optionally a pullulanase. In various embodiments
the produced maltotetraose composition has a DP4 concentration
higher than would result when saccharifying a whole ground corn
liquefact composition in place of the destructured starch-based
biomass composition; wherein the higher DP4 concentration is at
least 0.1%, 0.2%, 0.4%, or 0.5% higher.
[0245] In one embodiment the syrup is a composition comprising
maltopentaose (maltopentaose composition) produced by contacting
destructured starch-based biomass with a DP5 producing
alpha-amylase and optionally a pullulanase. In various embodiments
the produced maltopentaose composition has a DP5 concentration
higher than would result when saccharifying a whole ground corn
liquefact composition in place of the destructured starch-based
biomass composition; wherein the higher DP5 concentration is at
least 0.1%, 0.2%, 0.4%, 0.5%, 1.0%, 1.5%, or 2.0% higher.
[0246] In some embodiments the high force milled starch-based
biomass is processed for saccharification by mixing with an aqueous
solution to obtain a slurry. The aqueous solution may be obtained,
for example from water, thin stillage and/or backset. A
destructured starch-based biomass composition comprises
destructured starch-based biomass and optionally other components
such as the aqueous solution used to obtain a slurry. A slurry may
have dry solids (DS) of between 5-60%; 10-50%; 15-45%; 15-30%;
20-45%; 20-30% and also 25-40%.
[0247] Saccharification is often conducted as a batch process.
Saccharification typically is most effective at temperatures of
about 55.degree.-75.degree. C. and a pH of about 3.5-7.0, e.g., pH
5.0, preferably at a pH range of 4.0 to 6.0 and more preferably at
a pH range of 4.5 to 5.5. Saccharification may be performed, for
example, at a temperature between about 40.degree. C., about
55.degree. C., or about 65.degree. C. to about 70.degree. C., about
75.degree. C., or about 80.degree. C. In some embodiments, the
temperature is held between 45.degree. C. and 70.degree. C.; in
other embodiments, the temperature is held between 50.degree. C.
and 70.degree. C.; between 55.degree. C. and 70.degree. C.; between
60.degree. C. and 70.degree. C., between 60.degree. C. and
65.degree. C.; between 55.degree. C. and 65.degree. C. and between
55.degree. C. and 68.degree. C. In further embodiments, the
temperature is at least 45.degree. C., 48.degree. C., 50.degree.
C., 53.degree. C., 55.degree. C., 58.degree. C., 60.degree. C.,
63.degree. C., 65.degree. C. and 68.degree. C. In other
embodiments, the temperature is not greater than 65.degree. C.,
68.degree. C., 70.degree. C., 73.degree. C., 75.degree. C. and
80.degree. C.
[0248] In various embodiments the destructured starch-based biomass
is held in contact with at least one saccharifying enzyme for a
period of 5 minutes to 48 hours; or for a period of 5 minutes to 24
hours. In some embodiments the period of time is between 15 minutes
and 12 hours, 15 minutes and 6 hours, 15 minutes and 4 hours and
also 30 minutes and 2 hours.
[0249] Saccharification is normally conducted in stirred tanks,
which may take several hours to fill or empty. Enzymes typically
are added either at a fixed ratio to dried solids as the tanks are
filled or added as a single dose at the commencement of the filling
stage. A saccharification reaction to make a syrup typically is run
over about 24-72 hours, for example, 24-48 hours. When a maximum or
desired dextrose equivalents (DE) has been attained, the reaction
is stopped by heating to 85.degree. C. for 5 min., for example.
Further incubation will result in a lower DE, eventually to about
90 DE, as accumulated glucose re-polymerizes to isomaltose and/or
other reversion products via an enzymatic reversion reaction and/or
with the approach of thermodynamic equilibrium.
[0250] An effective concentration of enzyme used in the contacting
step for saccharification will vary according to the specific
process conditions and destructured starch-based biomass used.
Saccharification may be for any process such as to produce a syrup,
to produce fermentable sugars prior to fermentation, or to produce
fermentable sugars in a simultaneous saccharification and
fermentation process (SSF). Thus in some embodiments the contacting
step may occur prior to fermentation or during fermentation.
[0251] In some embodiments, the effective dose of a glucoamylase
for the contacting step will be in the range of 0.01 to 10 GAU/g
DS; also 0.05 to 10 GAU/g DS; also 0.1 to 5 GAU/g DS.
[0252] In some embodiments, alpha-amylase is included in the
contacting step. In some embodiments, the effective dose of
alpha-amylase for the contacting step will be in the range of 0.01
to 20 AAU/g DS, 0.01 to 10 AAU/g DS, 0.05 to 10.0 AAU/g DS, 0.5
to10.0 AAU/g DS, and also 0.5 to 2.0 AAU/g DS.
[0253] In various embodiments different types of alpha-amylase are
used including maltogenic alpha-amylase, DP4 producing
alpha-amylase, and DP5 producing alpha-amylase. In some
embodiments, the effective dose of maltogenic alpha-amylase to be
used in the contacting step will be in the range of 0.01 to 20 BU/g
DS, 0.01 to 15 BU/g DS, 0.05 to 15 BU/g DS, 0.5 to 10 BU/g DS. In
some embodiments, the effective dose of DP4 producing alpha-amylase
to be used in the contacting step will be in the range of 0.01-4
BMK/g DS, 0.01-2 BMK/g DS. In some embodiments, the effective dose
of DP3 producing alpha-amylase to be used in the contacting step
will be in the range of 0.01 to 10 SSU/g DS, 0.05-10 SSU/g DS, 0.5
to 10 SSU/g DS and 0.5 to 5 SSU/g DS. In some embodiments, the
effective dose of DP5 producing alpha-amylase to be used in the
contacting step will be in the range of 0.01-10 .mu.g/g ds, 0.05-10
.mu.g/g ds, 0.1-5 .mu.g/g DS.
[0254] In some embodiments, phytase is included in the contacting
step. In some embodiments, the effective dose of phytase to be used
in the contacting step will be in the range of 0.001 to 10 FTU/g
DS; also 0.005 to 10 FTU/g DS; and also 0.05 to 5 FTU/g DS. One
phytase unit (FTU) is the amount of enzyme, which liberates 1
micromole inorganic phosphorus per minute from sodium phytate,
0.0051 moles/liter, at 37.degree. C. and at pH 5.0.
[0255] In some embodiments, a protease is included in the
contacting step. In some embodiments, the effective dose of a
protease to be used in the contacting step will be in the range of
0.01 to 10 SAPU/g DS; 0.05 to 5 SAPU/g DS, and also 0.1 to 5 SAPU/g
DS. SAPU refers a spectrophotometric acid protease unit, wherein 1
SAPU is the amount of protease enzyme activity that liberates one
micromole of tyrosine per minute from a casein substrate under
conditions of the assay.
[0256] In some embodiments, a pullulanase is included in the
contacting step. In some embodiments, the effective dose of a
pullulanase to be used in the contacting step will be in the range
of 0.01 to 10 ASPU/g DS, also 0.05 to 10 ASPU/g DS; also 0.1 to 5
ASPU/g DS.
[0257] In some embodiments, a beta-amylase is included in the
contacting step. In some embodiments, the effective dose of a
beta-amylase to be used in the contacting step will be in the range
of 0.01 to 15 DP/g DS, 0.05 to 10 DP/g DS, and also 0.1 to 5 DP/g
DS.
[0258] In some embodiments, during the contacting step between
25-90% of the destructured starch-based biomass is solubilized to
produce oligosaccharides and monosaccharides. In some embodiments,
greater than 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%,
75%, 80%, 85% and 90% of the destructured starch is
solubilized.
[0259] After contacting the destructured starch-based biomass with
glucoamylase, and optionally with at least one of alpha-amylase and
phytase, for a period of time as indicated above, a soluble starch
substrate (mash) is obtained which comprises greater than 10%, 15%,
20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or 80%
glucose. The soluble starch substrate or mash is also referred to
herein as the glucose composition, or the composition comprising
fermentable sugars.
[0260] Prior to subjecting the mash including at least 10% glucose
to fermentation, the mash may be further exposed to an aqueous
solution comprising, for example, backset and/or corn steep (in the
case of corn grain) and adjusted to a pH in the range of pH 3.0 to
6.0; pH 3.5 to 5.5, or pH 4.0 to 5.5. In this embodiment of the
invention, the % DS of the mash may be diluted. For example, the DS
of the diluted mash maybe between 5 to 35%; 5 to 30%; 5 to 25%; 5
to 20%; 5 to 20%; 5 to 15%; and 5 to 10% less than the % DS of the
slurry in the contacting step. In one non-limiting example, if the
% DS of the slurry in the contacting step is approximately 32% and
the mash is further exposed to a diluting aqueous solution which
dilutes the DS between 5 to 10%, the DS of the mash to be fermented
will be between 22% and 27%. In some specific embodiments, if the
DS of the contacting slurry is between 30 to 35%, the DS of the
diluted slurry will be about 20 to 30%.
Fermentation
[0261] The composition comprising fermentable sugars (mash) may be
used in fermentation using a biocatalyst to produce a product,
herein called an End of Fermentation (EOF) product. An EOF product
may be any product made by the biocatalyst used in fermentation,
such as a metabolite that is naturally produced by the biocatalyst
or that the biocatalyst is genetically engineered to produce.
Examples of metabolite EOF products include but are not limited to
citric acid, lactic acid, succinic acid, monosodium glutamate,
gluconic acid, sodium gluconate, calcium gluconate, potassium
gluconate, glucono delta-lactone, sodium erythorbate, omega 3 fatty
acid, butanol, an amino acid, lysine, itaconic acid,
1,3-propanediol, and isoprene. In one embodiment, the EOF product
is ethanol.
[0262] Ethanologenic microorganisms include yeast, such as
Saccharomyces cerevisiae and bacteria, e.g., Zymomonas moblis,
expressing alcohol dehydrogenase and pyruvate decarboxylase. The
ethanologenic microorganism can express xylose reductase and
xylitol dehydrogenase, which convert xylose to xylulose. Improved
strains of ethanologenic microorganisms, which can withstand higher
temperatures, for example, are known in the art and can be used.
See Liu et al. (2011) Sheng Wu Gong Cheng Xue Bao 27(7): 1049-56.
Commercial sources of yeast include ETHANOL RED.RTM. (LeSaffre);
Thermosacc.RTM. (Lallemand); RED STAR.RTM. (Red Star); FERMIOL.RTM.
(DSM Specialties); and SUPERSTART.RTM. (Alltech).
[0263] Microorganisms that produce other metabolites, such as
citric acid and lactic acid, by fermentation are also known in the
art. See, e.g., Papagianni (2007) "Advances in citric acid
fermentation by Aspergillus niger: biochemical aspects, membrane
transport and modeling," Biotechnol. Adv. 25(3): 244-63; John et
al. (2009) "Direct lactic acid fermentation: focus on simultaneous
saccharification and lactic acid production," Biotechnol. Adv.
27(2): 145-52.
[0264] In one embodiment, the mash comprising at least 10% glucose
is subjected to fermentation processes using fermenting
microorganisms as described above. These fermentation processes are
described in The Alcohol Textbook 3.sup.rd ED, A Reference for the
Beverage, Fuel and Industrial Alcohol Industries, Eds Jacques et
al., (1999) Nottingham University Press, UK.
[0265] In some embodiments, the mash is fermented with a yeast at
temperatures in the range of 15 to 40.degree. C. and also 25 to
35.degree. C.; at a pH range of pH 3.0 to 6.5; also pH 3.0 to 6.0;
pH 3.0 to 5.5, pH 3.5 to 5.0 and also pH 3.5 to 4.5 for a period of
time of 12 to 240 hours, preferably 12 to 120 and more preferably
from 24 to 90 hours to produce an alcohol product, preferably
ethanol.
[0266] Yeast cells are generally supplied in amounts of 10.sup.4 to
10.sup.12, and preferably from 10.sup.7 to 10.sup.10 viable yeast
count per ml of fermentation broth. The fermentation will include
in addition to a fermenting microorganisms (e.g. yeast) nutrients,
optionally acid and additional enzymes.
[0267] In some embodiments, in addition to the raw materials
described above, fermentation media will contain supplements
including but not limited to vitamins (e.g. biotin, folic acid,
nicotinic acid, riboflavin), cofactors, and macro and
micro-nutrients and salts (e.g. (NH4).sub.2SO.sub.4;
K.sub.2HPO.sub.4; NaCl; MgSO.sub.4; H.sub.3BO.sub.3; ZnCl.sub.2;
and CaCl.sub.2).
[0268] In one embodiment, the contacting step is conducted in a
separate vessel from the fermenting step. It is also contemplated
that the contacting step and fermenting step may be conducted in a
simultaneous saccharification and fermentation (SSF) process in the
same vessel.
[0269] The SSF process may further comprise the addition of enzymes
to the process, including but not limited to: glucoamylase,
trehalase, pullulanase, isoamylase, hexokinase, xylanase, glucose
isomerase, xylose isomerase, phosphatase, phytase, .beta. amylase,
.alpha.-amylase, protease, cellulase, hemicellulase, lipase,
cutinase, isoamylase, redox enzyme, esterase, transferase,
pectinase, alpha-glucosidase, beta-glucosidase, lyase or other
hydrolases, or a combination thereof.
[0270] Additional enzymes that may be included in the fermentation
step may be the same or different from the enzymes used in the
contacting step. In some embodiments, the enzyme will include
alpha-amylases and glucoamylases, including destructured starch
hydrolyzing enzymes. In some embodiments, the glucoamylase and
alpha-amylase may occur in a blend. Some specific enzyme blends
include STARGEN.TM. 001 (DuPont Industrial Biosciences), which is a
blend of an alpha-amylase from A. kawachi and a glucoamylase from
A. niger. In some embodiments, the glucoamylase will be derived
from a Trichoderma glucoamylase, Humicola glucoamlyase, a Athelia
glucoamylase, a Talaromyces glucoamylase, a Aspergillus
glucoamylase, a Penicillium glucoamylase, a Trametes glucoamylase,
a Thermomyces glucoamylase and hybrid and variants glucoamylase
derived there from. In some preferred embodiments, the enzyme is
selected from a xylanase, a cellulase, a phytase and a
protease.
[0271] During fermentation for production of ethanol as the EOF
product the ethanol content of the broth or "beer" may reach about
8-18% v/v, e.g., 14-15% v/v. The broth may be distilled to produce
enriched, e.g., 96% pure, solutions of ethanol. Further, CO.sub.2
generated by fermentation may be collected with a CO.sub.2
scrubber, compressed, and marketed for other uses, e.g.,
carbonating beverage or dry ice production. Solid waste from the
fermentation process may be used as protein-rich products, e.g.,
livestock feed.
[0272] Continuous or fed-batch fermentation may be used. In a
fed-batch fermentation process, fermentation substrate is added in
increments as the fermentation progresses. Fed-batch systems are
useful when catabolite repression may inhibit the metabolism of the
cells and where it is desirable to have limited amounts of
substrate in the medium. The actual substrate concentration in
fed-batch systems is estimated by the changes of measurable factors
such as pH, dissolved oxygen and the partial pressure of waste
gases, such as CO.sub.2. Batch and fed-batch fermentations are
common and well known in the art.
[0273] Continuous fermentation is an open system where a defined
fermentation medium is added continuously to a bioreactor, and an
equal amount of conditioned medium is removed simultaneously for
processing. Continuous fermentation generally maintains the culture
at a constant high density where cells are primarily in log phase
growth. Continuous fermentation permits modulation of cell growth
and/or product concentration. For example, a limiting nutrient such
as the carbon source or nitrogen source is maintained at a fixed
rate and all other parameters are allowed to moderate. Because
growth is maintained at a steady state, cell loss due to medium
being drawn off should be balanced against the cell growth rate in
the fermentation. Methods of optimizing continuous fermentation
processes and maximizing the rate of product formation are well
known in the art of industrial microbiology.
[0274] In one embodiment the present destructured starch-based
biomass is used to produce an alcohol in SSF by contacting a slurry
comprising destructured starch-based biomass with an alpha-amylase,
a glucoamylase, a yeast and optionally an acid stable protease, at
a temperature below the starch gelatinization temperature of the
destructured starch-based biomass to produce oligosaccharides
fermentable by the yeast; and fermenting the oligosaccharides to
produce alcohol. In one embodiment the initial percentage of
alcohol production over the initial phase of SSF is a higher
percentage than would result when using a milled corn flour or
whole ground corn composition in place of the destructured
starch-based biomass composition. In one embodiment the initial
phase of SSF is the first 15 minutes of SSF. In one embodiment the
higher percentage of alcohol resulting in the initial phase of SSF
is a fold higher % selected from the list consisting of at least 2
fold higher % vs. corn flour; and at least 4 fold higher % vs.
whole ground corn. In one embodiment the end of SSF total alcohol
yield requires 3 fold less glucoamylase dosage as when using a
milled corn flour or whole ground corn composition in place of the
destructured starch-based biomass composition. In one embodiment up
to 10 fold less glucoamylase is required to produce a comparable
end of SSF alcohol yield for destructured starch-based biomass
composition than would be required for corn flour. In one
embodiment the alcohol is ethanol.
Recovery of Alcohol and Other End Products
[0275] One end product of the instant fermentation process is an
alcohol product, preferably ethanol. The end product produced
according to the process may be separated and/or purified from the
fermentation media. Methods for separation and purification are
known, for example by subjecting the media to extraction,
distillation and column chromatography. In some embodiments, the
end product is identified directly by submitting the media to
high-pressure liquid chromatography (HPLC) analysis.
[0276] In further embodiments, the fermentation broth may be
separated by for example centrifugation into the liquid phase and
solids phase and end products such as alcohol and solids recovered.
The alcohol may be recovered by means such as distillation and
molecular sieve dehydration or ultra filtration.
[0277] In some embodiments, the yield of ethanol will be greater
than 8%, 10%, 12%, 14%, 16% and 18% by volume. The ethanol obtained
according to process of the invention may be used as a fuel
ethanol, potable ethanol or industrial ethanol.
[0278] In further embodiments, the end product may include the
fermentation co-products such as distillers dried grains (DDG) and
distiller's dried grain plus solubles (DDGS), which may be used as
an animal feed.
Sugar Syrups
[0279] Maltose, the major sugar in maltose syrup, is widely used in
many industrial and consumers' applications, for example, as a
sweetener and in the preparation of maltitol, a low calorie
sweetener. Maltose is less sweet than high fructose corn syrup,
with a relative sweetness compared to sucrose of 30-40%. Maltose is
also used in brewing to increase the through-put and reduce haze
caused by varying quality of wort. Since maltose has a low freezing
point, high maltose syrup is useful in frozen desserts. Maltose
solutions exhibit lower viscosity and less humictantancy than equal
concentrations of glucose syrups and therefore is used a in candy
formulations to reduce stickiness. Maltitol is also used as a sugar
substitute, does not promote tooth decay, and has somewhat lesser
effect on blood sugar.
[0280] Maltotriose, maltotetrose and maltopentose are classified as
specialty syrups which are mainly used in the food industry.
EXAMPLES
[0281] The present invention is further defined in the following
Examples. It should be understood that these Examples, while
indicating preferred embodiments of the invention, are given by way
of illustration only. From the above discussion and these Examples,
one skilled in the art can ascertain the essential characteristics
of this invention, and without departing from the spirit and scope
thereof, can make various changes and modifications of the
invention to adapt it to various uses and conditions.
[0282] The meaning of abbreviations is as follows: "h" or "hr"
means hour(s), "min" means minute(s), "s" or "sec" means second(s),
"d" means day(s), "L" means liter(s), "mL" means milliliter(s),
".mu.L" means microliter(s), "kg" means kilograms, "g" means grams,
".mu.g" means microgram(s), "ng" means nanogram(s), "mg" means
milligrams, "g/L" means grams per liter, "mM" means millimolar,
".mu.M" means micromolar, "nm" means nanometer(s), "mm" means
millimeter, "cm" means centimeter(s), ".mu.mol" means micromole(s),
"pmol" means picomole(s), "N" means newton(s), "G-force" means
force of gravity, "MPa" means megapascals, "kPa" means kilopascals,
"psi" means pounds per square inch, "CF" means crystalline
fraction, "CDS" means coherent domain size, "F" means force, "''"
or "in" means inch(s), DS is dry solids; GAU is Glucoamylase Units;
BU is units of OPTIMALT.RTM. 2G activity; DE is Dextrose
Equivalent(s); AAU is alpha amylase activity units; gds means grams
of dry solids; MT=metric ton; H means hour(s); DP is activity units
of OPTIMALT.RTM. BBA; ASPU is activity units of OPTIMAX.RTM.
L-1000; SSU is activity units of fungal alpha-amylase; BMK is
activity units of OPTIMALT.RTM. 4G; % HS is % higher sugars,
meaning DP4 and higher DPs (DP5, DP6 etc.); SAPU is units of
protease activity; AAU is units of bacterial alpha amylase
activity, FTU is units of phytase activity, SAPU refers a
spectrophotometric acid protease unit.
General Methods
Non High Force Milling
[0283] Milling was performed on the whole grains: wheat, sorghum,
rice, and vacuum dried cassava to generate control materials for
XRD, particle size, and fermentation using a model ZM 200 12
toothed rotor mill (Retsch) and a 120 micron screen. The mill was
run at 12000 rpm. Samples were milled in 200 g batches.
[0284] Course ground corn is hammer milled corn.
[0285] Fine ground corn is purchased flour (Azure Farm).
Centrifugal Ring and Puck Milling or High Force Milling
[0286] High starch content biomass was treated in a vibratory
pulverizer (model VP-1989: 3 phase Vibratory Pulverizer; Bico Inc.,
Burbank, Calif.); also called a ring and puck mill. The pulverizer
consists of a hardened chrome alloy steel bowl (8.125'' (20.64 cm)
inner diameter), which contains two rings of 1/2'' (1.27 cm) thick
hardened steel, one with an inner diameter of 3.06'' (7.77 cm) and
the other with an inner diameter of 2.05'' (5.21 cm) and a solid
puck (i.e. disc) with diameter of 1.64'' (4.17 cm). The lengths of
the rings and puck and corresponding depth of the bowl are 2''
(5.08 cm). The bowl containing the rings, puck, and sample were
vibrated with a one horsepower eccentric motor at 900 rpm. The
radius of eccentricity of the mill is 4.5 mm, which is the radius
of the circle that is drawn out by the vibration of the mill. The
power of the mill was determined by the using the measured current,
applied voltage, and a power factor of 0.8 in the standard equation
for a three phase motor.
Moisture Assay
[0287] The moisture content of the biomass was measured using a
Mettler-Toledo HR73 Halogen Moisture Analyzer. An amount of at
least 0.5 g (but less than 3 g) of starch based feedstock was used
for each measurement. Each sample was weighed out into an aluminum
pan and heated according to the standard drying program to at
temperature of 105.degree. C. Each sample was held at 105.degree.
C. until the measured mass loss was <1 mg/50 s, at which point
the weight was recorded and the moisture content determined by the
ratio of the difference between the initial and final weights to
the initial weight of the starch based feedstocks.
Particle Size Assay
[0288] Particle size distribution (PSD) was determined via laser
diffraction (reference ISO 13320-1:1999) using a Malvern
Mastersizer 2000 (Malvern Instruments, Malvern UK). Sample
particles were dispersed in air using a Malvern Scirocco 2000 dry
disperser unit with the dispersion jet pressure set to about 60 psi
(0.41 MPa) and the feed rate set to about 45% of maximum. The
dispersed particles were pneumatically conveyed through the
Mastersizer's flow cell, which was outfitted with quartz windows
for optical access. Laser light entering the flow cell was
diffracted by the particles, and the diffraction pattern was imaged
onto an array of detectors. The PSD was calculated by analyzing the
recorded diffraction pattern using the Fraunhofer scattering model
(described in ISO 13320-1:1999, Annex A). A standard reference for
PSD measurement and terminology is T. Allen, Particle Size
Measurement, Vol. 1, 5.sup.th Ed. (Chapman & Hall 1997).
XRD Technique and Analysis for Crystallinity Assessment
[0289] X-ray diffraction data were obtained with a Philips X'PERT
automated powder diffractometer, Model 3040. The diffractometer is
equipped with automatic variable anti-scatter and divergence slits,
X'Celerator RTMS detector, and Ni filter. The radiation is
CuK(alpha) (45 kV, 40 mA). Data were collected at room temperature
from 4 to 80 degrees 2-theta; using a continuous scan with an
equivalent step size of 0.02 degrees; and a count time of 80
seconds per step in theta-theta geometry. Samples were packed into
an aluminum sample holder and run with no additional grinding.
MDI/Jade software version 9.1 was used to convert the data to text
format for further processing. Microsoft.RTM. Excel.RTM. (2010)
software was used for all additional processing.
[0290] The degree of crystallinity is calculated from the X-ray
diffraction data by using a crystalline area integration method
based on Cheetham and Leping (Carbohydrate Polymers 36:277-284
(1998)), Nara et al. (Starch 35,12:407-410 (1983)), and Benedetti
et al. (Journal of Material Science 18.4:1039-1048 (1983)). The
intensities are first normalized over a limited range of data
(10-30 2-theta). The normalization occurs by creating a baseline
connecting the upper and lower bounds of 10 and 30 2-theta and then
dividing the intensities by the integrated area below the
intensities curve and above the baseline. After normalization, the
data is smoothed by using a Savitzky-Golay filter. The crystalline
and amorphous regions are then separated by a function which
connects peak baselines. The upper region is the crystalline
portion, and the lower region is the amorphous portion. The
crystalline portion area and the total diffraction area are
integrated. The degree of crystallinity is taken to be the ratio of
the crystalline area over the total diffraction area.
Degree of Crystallinity = Crystalline Area Total Diffraction Area
##EQU00003##
Sugar Composition and Concentration Analysis by HPLC
[0291] 166.67 .mu.L of sample was diluted in 1.5 mL ultrapure water
and incubated in a thermo-mixer at 99.degree. C. for 15 minutes.
Diluted samples were cooled and then filtered using a 96-well
filtration plate (Millipore Multiscreen HTS, 0.22 .mu.m Durapore
membrane) prior to HPLC analysis. [0292] RHM column: Phenomenex
#00A0132K0 s/n 356583222 300.times.7.8 mm; Column [0293]
Temperature: 85.degree. C.; Mobile Phase: DI H.sub.2O HPLC grade;
Flow Rate: 0.6 ml/min; [0294] Detector: RI detection; Detector
Temperature: 40.degree. C.; Injection Volume: 1 .mu.L
[0295] A designation of DP1 is a monosaccharide, which is
substantially glucose; a designation of DP2 is a disaccharide,
which is substantially maltose and may include trehalose; a
designation of DP3 is a trisaccharide, which is substantially
maltotriose; a designation of DP4 is a tetrasaccharide, which is
substantially maltotetraose; and the designation "DPn" refers to
those components which elute together, containing among others an
oligosaccharide having a degree of polymerization (DP) of 5 or
greater, but also components like sulfate, chloride, and non-starch
polysaccharides.
[0296] Area percentages of the different sugars (DPn, DP4, DP3,
DP2, and DP1) were calculated by dividing the area of that specific
sugar peak by the area of all five peaks. The combined area
percentages of DPn, DP4, DP3, DP2 and DP1 add up to 100%.
Concentrations of the different sugars (DP3, DP2, and DP1) were
calculated by multiplying the peak area of that specific sugar with
the response factor, which is determined by linear regression of
known concentrations of the specific sugar (y-axis) and the
obtained peak areas (x-axis).
Preparation of Reference Corn Liquefact
[0297] Corn kernels were ground with a hammer mill with a 2 mm
sieve at 10,000 rpm. The resulting corn meal was mixed with water
producing a slurry with up to 30% DS and the pH was adjusted to 5.5
with 2 M H.sub.2SO.sub.4. 0.155 kg/MT SPEZYME.RTM. CL was added and
the mixture was kept under constant stirring at 60.degree. C. for
30 minutes before being brought to 85.degree. C. for 90 minutes.
Then the mixture slurry was cooled down to room temperature and the
pH was brought to 4.5 with 2 M H.sub.2SO.sub.4 resulting in a corn
liquefact with 31.6% DS.
Enzymes
[0298] A fungal alpha-amylase from Aspergillus clavatus having an
activity of 301 U/mg on amylopectin, and optimal activity at
temperature of 66.degree. C. [0299] A bacterial alpha-amylase from
Cytophaga sp. having an activity of 2459 U/mg on amylopectin.
[0300] A fungal gluco-amylase from Aspergillus fumigatus (AfGA)
having an activity of 242.9 U/mg on DP7. [0301] A fungal
alpha-amylase from Aspergillus kawachii (AkAA) having an activity
of 58.9 U/mg on amylopectin. This enzyme is commercially available
as GC626 from Genencor/Danisco/Dupont. [0302] Acid stable fungal
gluco-amylase from Trichoderma reesei (TrGA) which is described in
U.S. Pat. No. 7,037,704. [0303] The following enzymes are
commercially available from Genencor/Danisco/Dupont: [0304]
OPTIMAX.RTM. 4060 VHP [0305] OPTIMAX.RTM. HDS [0306] OPTIMALT.RTM.
2G [0307] SPEZYME.RTM. XTRA [0308] OPTIMALT.RTM. BBA [0309]
OPTIMAX.RTM. L-1000 [0310] OPTIMALT.RTM. 4G [0311] SPEZYME.RTM. RSL
[0312] FERMGEN.TM. 2.5x [0313] SPEZYME.RTM. CL
Dextrose Equivalents Measurement
[0314] The term "DE," or "dextrose equivalent," is defined as the
percentage of reducing sugar, i.e., D-glucose, as a fraction of
total carbohydrate in a syrup. Dextrose equivalents (DE) were
measured using the Schoorl method (Schoorl, N., Zurjodometrischen
Zukerbestimmung mittles Fehlingscher Losung., Zeitschr. F. agnew.
Chem., 12, 633 (1899); Hodge, J. E & Davis, H. A. Selected
Methods for Determining Reducing Sugars. United States Department
of Agriculture Technical Bulletin A1C333. (1952); Schenck, F. W.
& Hebeda, R. E., Starch Hydrolysis Products, Worldwide
Technology Production and Applications, p 379, VCH Publishers
(1992))
True Solubility
[0315] True solubility is the actual solubility in a dry sample
which is calculated from the measured solubility through RI.
[0316] Determination of the actual solubility in a dry sample which
is calculated from the measured solubility through RI using the
following formula:
Y=0.0046720x 2+0.4925651x+3.8924217
[0317] Where Y is the true soluble and x is the solubility
determined with RI.
[0318] This is due to the remaining starch not participating in the
RI determination. This diminishes as greater solubility is
achieved.
[0319] The solubility as obtained from the described procedure can
then be inserted as x in the equation extracted from the graph
below.
EXAMPLE
[0320] Start material: 28% ds (starch)
[0321] RI.sub.sup: 1.3784=>equals 29.49% ds retrieved from the
DE 95 conversion table.
29.49% ds/(28*1.1)*100=95.7
[0322] RI.sub.total: 1.3836=>equals 32.45% ds retrieved from DE
95.7 conversion table
% starch solubility=29.49/32.45=90.9
[0323] Corrected % starch solubility:
0.0046720*90.9.sup.2+0.4925651*90.9+3.8924217=87.3
Oil Analysis
[0324] A sample of 20 g in 50 ml centrifuge tube is heated to
80.degree. C. for 30 min. [0325] The tube is then centrifuged at
10000 rpm for 2 min. [0326] 2 ml of hexane is added carefully to
the top of the centrifuged sample and gently swirled, then slowly
shaken for 5 min. [0327] Let the tubes sit for 15 min total. [0328]
200 .mu.l of the oil containing hexane is siphoned off and
centrifuged to break any emulsion. [0329] The sample is then
diluted further with hexane and 10 .mu.l is injected into the LC.
[0330] This is free oil. [0331] Then take the hexane containing
tubes and shake them vigorously for a few minutes and
re-centrifuge. [0332] This recovers the total oil.
Example 1
Destructuring in a Centrifugal Force Ring-Roller Mill
[0333] Centrifugal force ring-roller mills (cfRRMs) have been
described in Perry's Chemical Engineers' Handbook (8.sup.th
Edition, ch 21 p 60), and can contain a wide variety of dimensions
based on the scale of the operation. Dimensions for representative
scales of cfRRMs were chosen and the forces and stresses generated
for these examples of commercial scale cfRRMs were calculated based
on their dimensions and masses. The dimensions, masses, and results
are given in Table 1. The mill designs have at least two rollers,
each attached to a swinging arm, with the arms attached to a drive
shaft. The rollers revolve within a chamber, which causes the
rollers to press radially outward against a ring.
TABLE-US-00001 TABLE 1 Centrifugal Force Ring-Roller Mill
Specifications and Forces Parameter cfRRM-1 cfRRM-2 cfRRM-3 cfRRM-4
RPM 90 150 100 100 Chamber Diameter [in] 40 40 80 100 Roller
Diameter [in] 13.3 13.3 26.7 33.3 Roller/Ring Height [in] 10 10 20
29.2 Roller Density [g/cm3] 8 8 8 8 Mass of Roller [kg] 183 183
1,464 3,337 Centrifugal Force [N] 5512 15311 108,770 309,810
Hertzian Stress (w/o 8140 13566 18,046 22,557 material) [psi] G
Force 3.07 8.53 7.57 9.47
[0334] Equation 1 was used to calculate the centrifugal forces in
each apparatus. The center-of-mass radii for the rollers were found
by first setting the axis along the central drive shaft as the
origin. The distances from the center of each roller to the central
drive shaft are the center-of-mass radii. They are positioned so
that they are against the ring, as well as equidistant from each
other along the circumference of the ring (such that the center of
mass of the all of the rollers considered together, would align
with the central shaft on which they are attached).
[0335] The "g-forces" in the centrifugal force roller mill were
calculated by dividing the centripetal acceleration (i.e. F/m) by
the acceleration owing to gravity (.about.9.8 m/s.sup.2).
[0336] Thus biomass treated in the described apparatuses would be
subjected to forces of >5000 N and "G-forces" of .about.3 G, or
forces >15,000 N and "G-forces" <9 G, or forces of >100,00
N and "G-forces" of <8 G, or forces >300,000 N and "G-forces"
<10 G.
[0337] Equation 3 was used to calculate Hertzian stress. The number
used for poison's ratio (v) was 0.3 and the number used for elastic
modulus (E) was 2.1*10.sup.11 Pa. These values were chosen based on
assumptions that are generally applied to common metals. These same
numbers were used for v.sub.1 as well as v.sub.2, and for E.sub.1
as well as E.sub.2. The centrifugal forces calculated and given in
Table 1 were used as F. The Ring/Roller Heights given in Table 1
were used as I. For purposes of the calculation, an assumption was
made that the roller and ring surfaces were perfect cylinders,
while in reality the roller becomes concave and does not present an
even line contact with the ring.
[0338] For a combined milling system (mill and auxiliary equipment
such as blower or classifier), the hp can vary dramatically based
on the size of the milling system. The full power capabilities for
typical cfRRM systems can be anywhere from .about.20 hp up to
<5000 hp. Once the mill is operated in the desired force and
stress range (>5000 N and >5,000 psi (34.47 MPa)), then the
air handling system and classifier are modified to achieve a
throughput such that the power draw on the machine divided by the
throughput yields a specific energy that is <40% of the total
combustible energy of the feed material. All of these running
modifications are routine to one of skill in the art. The
starch-based biomass product is expected to have crystallinity that
is reduced, as compared to the crystallinity of the starch-based
biomass which is not treated, by at least about 25% and efficient
saccharification without using a heating step. In the case of corn
kernels the specific energy input is expected to be <7,200
kJ/kg, which is <40% of the combustible energy of corn kernel
biomass (.about.18,000 to 20,000 kJ/kg of biomass).
Example 2
Destructuring Effect of Ring and Puck Milling of Whole Corn
Kernels
[0339] 5 g of whole corn kernels (11.1% moisture) were divided
equally in the three spaces between the media in the bowl (i.e.
between the bowl and larger ring, between the larger ring and
smaller ring, and between the smaller ring and puck) of the
vibratory pulverizer (ring and puck mill) described in General
Methods for centrifugal milling. The ring and puck mill was run for
5 min. The throughput for the milling was .about.1 g/min or
1.67.times.10.sup.-5 kg/s, which gives a specific energy input of
0.134 hp/(1.67.times.10.sup.-5 kg/s) or .about.1.68 kWh/kg or
.about.6.03 kJ/g.
[0340] The corn kernels were subjected to forces ranging from 646
to 1,511 N and "G-forces" ranging from 21.5 to 32.3 as calculated
and described below. [0341] Dimensions and mass of the media in the
ring and puck mill are: [0342] Bowl: 8.125'' (20.64 cm) inner
diameter, 2'' (5.08 cm) depth, 18.80 lbs (8.53 kg) [0343] Large
Ring: 6.125'' (15.56 cm) inner diameter, 2'' (5.08 cm) depth, 0.6''
(1.52 cm) thickness, 6.60 lbs (2.99 kg) [0344] Small Ring: 4.1''
(10.41 cm) inner diameter, 2'' (5.08 cm) depth, 0.6'' (1.52 cm)
thickness, 4.69 lbs (2.13 kg) [0345] Puck: 3.270'' (8.31 cm)
diameter, 2'' (5.08 cm) depth, 4.49 lbs (2.04 kg) The ring and puck
mill operates at a constant 900 rpm angular frequency. The circle
of eccentricity of the motor (i.e. the circumference of the circle
drawn out by the motion of the oscillating stage) is 1.11''.
Equation 1 can be used to calculate the centrifugal force at the
interface between any of the media in the mill. The center-of-mass
radius is calculated according to Equation 7.
[0345] r = i n m i r i m total Equation 7 ##EQU00004##
where n is the number of media, i is the specific index identifying
each unique medium, m.sub.i is the specific mass of an indexed
medium, r.sub.i is the specific center-of-mass radius of an indexed
medium, and m.sub.tot is the total or combined mass of all the
media. The total mass of the medial is 15.78 lbs (7.16 kg). The
center-of-mass radii of the media are found first by setting the
center of the milling stage as the origin. Next, the large ring,
small ring, and puck are placed into the bowl and pushed to one
side, causing the center-of-mass radius for each medium to be
offset from the center of the milling stage, as well as the bowl.
The circle of eccentricity of the motor (i.e. the circumference of
the circle drawn out by the motion of the oscillating stage) is
1.11'', which further offsets the center of mass from the center of
the milling stage by the radius of the circle (0.18''). The
specific center-of-mass radius, r.sub.i, for each medium is the
distance from the bowl center to the center of each medium, with
the addition of 0.18'' to account for the motion of the stage. For
example, the center-of-mass radius of the puck is 1.40'' (5.44 cm),
which is found by subtracting the large ring thickness (0.6''; 1.52
cm), small ring thickness (0.6''; 1.52 cm), and puck radius
(1.64'') from the bowl inner radius (4.0625'') and adding 0.18''.
Lastly, the "g-forces" in the ring and puck mill can be calculated
by dividing the centripetal acceleration (i.e. F/m) by the
acceleration due to gravity (.about.9.8 m/s.sup.2). Table 2
summarizes the masses, center-of-mass radii, centrifugal forces at
the interfaces between the media, and the "g-forces" in the ring
and puck mill.
TABLE-US-00002 TABLE 2 Parameters of the apparatus used for
centrifugal milling Mass Center-of-mass Force "G- Pressure
Interface [kg] radius [m] [N] force" [psi] Bowl-Large Ring 7.16
0.024 1,511 21.5 4,934 Large Ring-Small Ring 4.17 0.030 1,121 27.4
5,843 Small Ring-Puck 2.04 0.036 646 32.3 6923
After milling, the whole corn kernels were reduced to a fine powder
with a d.sub.50 of .about.27 .mu.m. The PSD is shown in FIG.
3A.
[0346] A comparison was made with conventional hammer milled corn
kernels which showed a d.sub.50 of 543.2 .mu.m, as well as a
biomodal distribution (FIG. 3B). The XRD patterns indicating the
destructured nature of the starch granules in corn kernels that
were conventionally processed to corn flour or ring and puck milled
are shown in FIG. 4. The crystallinity of the ring and puck milled
corn kernels, determined as described in General Methods, decreased
from 24.4% to 0.9%. Thus the crystalline features that are present
for hammer milled whole corn kernels were virtually eliminated in
the ring and puck milled sample, indicating destructuring of the
whole corn kernels. The ring and puck milling process described in
this example was performed multiple times to generate enough
destructured material for use in later examples. The material is
called high force milled corn or ring and puck milled corn.
Example 3
Destructuring Effect of Ring and Puck Milling Whole White Wheat
Kernels
[0347] 5 g of whole white wheat kernels (0.84% moisture) were
divided equally in the three spaces between the media in the bowl
(i.e. between the bowl and larger ring, between the larger ring and
smaller ring, and between the smaller ring and puck) of the
vibratory pulverizer (ring and puck mill) described in General
Methods for centrifugal milling. The ring and puck mill was run for
5 min. The throughput for the milling was .about.1 g/min or
1.67.times.10.sup.-5 kg/s, which gives a specific energy input of
0.134 hp/(1.67.times.10.sup.-5 kg/s) or .about.1.68 kWh/kg or
.about.6.03 kJ/g.
[0348] After milling, the whole white wheat kernels were reduced to
a fine powder with a d.sub.50 of 17.7 .mu.m. The PSD is shown in
FIG. 5A. The corresponding XRD pattern indicating the destructured
nature of the starch granules is shown in FIG. 5B. The
crystallinity, determined as described in General Methods,
decreased from 12.9% to 0.1%. Control wheat kernels were gently
crushed with a mortar and pestle into a fine powder, and used for
XRD measurement (FIG. 5B). The crystalline features that are
present for whole white wheat kernels crushed with a mortar and
pestle were virtually eliminated after ring and puck milling. The
milling process described in this example was performed multiple
times to generate enough destructured material for use in later
examples.
Example 4
Destructuring Effect of Ring and Puck Milling Whole Sorghum
[0349] 5 g of whole sorghum (2.04% moisture) were divided equally
in the three spaces between the media in the bowl (i.e. between the
bowl and larger ring, between the larger ring and smaller ring, and
between the smaller ring and puck) of the vibratory pulverizer
(ring and puck mill) described in General Methods for centrifugal
milling. The ring and puck mill was run for 5 min. The throughput
for the milling was .about.1 g/min or 1.67.times.10.sup.-5 kg/s,
which gives a specific energy input of 0.134
hp/(1.67.times.10.sup.-5 kg/s) or .about.1.68 kWh/kg or .about.6.03
kJ/g.
[0350] After milling, the whole sorghum was reduced to a fine
powder with a d.sub.50 of 17.4 .mu.m. The PSD is shown in FIG. 6A.
The corresponding XRD pattern indicating the destructured nature of
the starch granules is shown in FIG. 6B. The crystallinity,
determined as described in General Methods, decreased from 28.0% to
0.2%. Control whole sorghum was gently crushed with a mortar and
pestle into a fine powder, and used for XRD measurement (FIG. 6B).
The crystalline features that are present for whole sorghum crushed
with a mortar and pestle were virtually eliminated after ring and
puck milling. The milling process described in this example was
performed multiple times to generate enough destructured material
for use in later examples.
Example 5
Destructuring Effect of Ring and Puck Milling Whole Brown Rice
[0351] 5 g of whole brown rice (1.42% moisture) were divided
equally in the three spaces between the media in the bowl (i.e.
between the bowl and larger ring, between the larger ring and
smaller ring, and between the smaller ring and puck) of the
vibratory pulverizer (ring and puck mill) described in General
Methods for centrifugal milling. The ring and puck mill was run for
5 min. The throughput for the milling was .about.1 g/min or
1.67.times.10.sup.-5 kg/s, which gives a specific energy input of
0.134 hp/(1.67.times.10.sup.-5 kg/s) or .about.1.68 kWh/kg or
.about.6.03 kJ/g.
[0352] After milling, the whole brown rice was reduced to a fine
powder with a d.sub.50 of 11.5 .mu.m. The PSD is shown in FIG. 7A.
The corresponding XRD pattern indicating the destructured nature of
the starch granules is shown in FIG. 7B. The crystallinity,
determined as described in General Methods, decreased from 28.6% to
0.1%. Control whole brown rice was gently crushed with a mortar and
pestle into a fine powder, and used for XRD measurement (FIG. 7B).
The crystalline features that are present for whole brown rice
crushed with a mortar and pestle were virtually eliminated after
ring and puck milling. The milling process described in this
example was performed multiple times to generate enough
destructured material for use in later examples.
Example 6
Destructuring Effect of Ring and Puck Milling Cassava
[0353] Frozen cassava pieces were cut into .about.1/2'' cubes and
vacuum dried for 4 hours at 45.degree. C. 5 g of dried cassava
(7.05% moisture) were divided equally in the three spaces between
the media in the bowl (i.e. between the bowl and larger ring,
between the larger ring and smaller ring, and between the smaller
ring and puck) of the vibratory pulverizer (ring and puck mill)
described in General Methods for centrifugal milling. The ring and
puck mill was run for 5 min. The throughput for the milling was
.about.1 g/min or 1.67.times.10.sup.-5 kg/s, which gives a specific
energy input of 0.134 hp/(1.67.times.10.sup.-5 kg/s) or .about.1.68
kWh/kg or .about.6.03 kJ/g.
[0354] After milling, the dried cassava pieces were reduced to a
fine powder with a d.sub.50 of .about.14.0 .mu.m. The PSD is shown
in FIG. 8A. The corresponding XRD pattern indicating the
destructured nature of the starch granules is shown in FIG. 8B. The
crystallinity decreased from 48.4% to 4.0%. Control dried cassava
pieces were gently crushed with a mortar and pestle into a fine
powder, and used for XRD measurement (FIG. 8B). The crystalline
features that are present for dried cassava crushed with a mortar
and pestle, determined as described in General Methods, were
virtually eliminated after ring and puck milling. The milling
process described in this example was performed multiple times to
generate enough destructured material for use in later
examples.
Example 7
Extent of Destructuring with Processing Time in Ring and Puck
Mill
[0355] Approximately 5 g of whole corn kernels were ring and puck
milled in the same manner as described in Example 2 except that the
milling time was varied to generate corn with different levels of
destructuring. It can be seen from FIG. 9 that the material was
significantly destructured after only 30 seconds of milling. The
crystallinity for each time sample was determined (see General
Methods) giving results as follows: 30 s: 6.92%, 1 min: 5.22%, 2
min: 2.49%, 3 min: 1.91%, and 5 min: 0.86%. Corn flour (not ring
and puck milled), with a crystallinity of about 26% in this
experiment, is shown for reference.
[0356] The amount of energy used to produce each time sample was
calculated by dividing the steady state power output [kW] of the
apparatus used by the throughput [g/s]. Based on this calculation,
the percent of the energy content of the corn used to produce the
destructured sample was calculated for a corn energy value of
18,000 or 20,000 kj/kg and is shown in Table 3.
TABLE-US-00003 TABLE 3 Processing energy for destructured
starch-based biomass samples. % of corn energy Time [s] kJ/kg
18,000 20,000 30 603 3% 3% 60 1206 7% 6% 120 2412 13% 12% 180 3618
20% 18% 300 6030 34% 30%
Example 8
Glucose Production from High Force Milled Corn using
Gluco-Amylases
[0357] In this experiment two commercial enzyme products were
tested to see whether glucose syrup can be made directly from high
force milled corn without application of liquefaction with
alpha-amylase. The results were compared to those obtained with a
traditional corn liquefact.
[0358] A 10% DS aqueous slurry of high force milled corn kernels,
treated as described in Example 2, was incubated at pH 4.4 and
60.degree. C. with 0.16 GAU gds OPTIMAX.RTM. 4060 VHP (0.16
activity units are added per gram dry solids) or 0.10 GAU gds
OPTIMAX.RTM. HDS.
[0359] As reference, corn liquefact prepared as described in
General Methods was diluted to 10% DS and the slurry was also
incubated at pH 4.4 at 60.degree. C. with 0.16 GAU gds OPTIMAX.RTM.
4060 VHP, or 0.10 GAU gds OPTIMAX.RTM. HDS.
[0360] Each slurry was constantly shaken (orbital shaker incubator;
100 rpm) and samples were taken at different time intervals (t) for
determination of sugar composition and concentration by HPLC (see
General Methods). The results are shown in Table 4.
TABLE-US-00004 TABLE 4 Sugar compositions of high force milled corn
and corn liquefact treated with different gluco-amylases t DPn DP4
DP3 DP2 DP1 DP3 DP2 DP1 enzyme substrate [h] % % % % % (% w/V) (%
w/V) (% w/V) OPTIMAX .RTM. High 2 60.39 0.62 1.21 1.31 34.40 0.09
0.10 2.51 4060 VHP force 4 36.95 0.51 1.38 3.23 55.80 0.11 0.27
4.30 0.16 GAU milled 6 22.97 0.34 0.74 4.20 69.51 0.06 0.36 5.52
gds corn 20 5.25 0.54 0.55 2.32 88.80 0.05 0.22 7.70 25 4.43 0.50
0.41 2.25 89.91 0.04 0.22 8.00 30 4.19 0.49 0.56 2.07 90.33 0.05
0.20 7.92 45 3.87 0.47 0.59 2.04 90.58 0.06 0.20 8.09 50 4.04 0.42
0.59 2.03 90.37 0.06 0.20 8.10 Whole 2 31.88 1.61 7.46 13.63 44.55
0.54 1.00 3.02 ground 4 20.03 0.75 1.82 16.39 60.09 0.14 1.25 4.21
corn 6 13.37 1.01 1.19 12.21 71.04 0.09 0.93 4.96 liquefact 20 4.01
0.58 1.20 2.42 90.25 0.10 0.20 6.93 25 3.82 0.61 1.21 2.28 90.56
0.10 0.19 7.06 30 3.65 0.52 1.17 2.16 91.22 0.10 0.18 7.13 45 3.49
0.58 1.31 2.31 91.41 0.12 0.21 7.73 50 3.48 0.53 1.36 2.27 90.79
0.11 0.19 7.11 OPTIMAX .RTM. High 2 72.10 0.51 1.01 0.68 23.60 0.07
0.05 1.60 HDS force 4 53.49 0.59 1.45 1.85 40.56 0.12 0.15 3.08
0.10 GAU milled 6 36.23 0.45 1.43 3.40 56.10 0.12 0.29 4.36 gds
corn 20 7.22 0.38 0.49 2.86 86.52 0.05 0.27 7.38 25 6.07 0.38 0.49
2.29 88.32 0.05 0.22 7.91 30 4.52 0.31 0.43 1.90 90.47 0.04 0.18
7.89 45 4.43 0.24 0.60 1.36 91.04 0.06 0.13 8.09 50 4.24 0.14 0.45
1.31 91.52 0.04 0.13 8.02 Whole 2 46.51 3.93 10.15 8.96 29.63 0.74
0.66 2.01 ground 4 31.74 1.75 7.93 13.55 44.10 0.58 1.00 3.01 corn
6 19.75 0.87 3.50 17.23 57.76 0.26 1.30 4.02 liquefact 20 4.88 0.85
1.18 6.50 85.07 0.09 0.53 6.37 25 4.36 0.77 1.21 4.91 87.81 0.10
0.40 6.66 30 3.94 0.70 1.15 3.91 88.85 0.10 0.35 7.44 45 3.48 0.56
1.16 2.74 91.25 0.10 0.24 7.23 50 3.50 0.45 1.17 2.52 90.89 0.10
0.22 7.14
[0361] Table 4 shows that saccharification of high force milled
corn with OPTIMAX.RTM. 4060 VHP resulted in similar DP1 percentages
as incubations with whole ground corn liquefact. For the high force
milled corn the percentage of DPn was found to be higher, while DP4
and DP3 and DP2 were lower than the incubations with whole ground
corn liquefact. The lower concentration of DP3 can be explained by
limited exposure to alpha-amylase activity in the incubated high
force milled corn, as this material has not been liquefied with
enzymes like the reference liquefact.
[0362] The concentration of DP1 was found to be higher for the high
force mill treated corn when compared to whole ground corn
liquefact. This was also the case for the incubations with
OPTIMAX.RTM. HDS. The results indicate that the high force mill
treatment resulted in higher solubilisation of starch (shown in
higher initial DPn %) than with traditional liquefaction treatment,
meaning that more substrate was available for glucose production,
resulting in higher concentrations of glucose.
[0363] The DP2 for the OPTIMAX.RTM. HDS incubation with high force
milled corn was lower contributing to the higher concentration of
DP1 when compared with the whole ground corn liquefact. This could
also be related to limited exposure to alpha-amylase activity.
[0364] This example shows that glucose syrups were produced from
corn without a liquefaction step by treatment of the corn with high
force milling and incubating this material with gluco-amylase
containing enzyme products. The high force mill treated corn
resulted in highly soluble starch with a low DE resulting in higher
glucose yield than possible with a conventional corn liquefact
process. Thus high force milling resulted in a better substrate for
glucose syrup production than can be obtained via traditional
liquefaction.
Example 9
Maltose Production from High Force Milled Corn using a Maltogenic
Alpha Amylase
[0365] In this experiment a maltogenic alpha-amylase with and
without additional alpha-amylase was tested to see whether maltose
syrup can be made directly from high force milled corn without
application of liquefaction with alpha-amylase. The results were
compared to those obtained with a traditional corn liquefact.
[0366] A 10% DS aqueous slurry of high force milled corn (prepared
as described in Example 2) was incubated at pH 5.0 and 60.degree.
C. with 8 BU gds OPTIMALT.RTM. 2G with or without 0.5 AAU gds
SPEZYME.RTM. XTRA.
[0367] As reference, corn liquefact (General Methods) was diluted
to 10% DS and the slurry was also incubated at pH 4.4 at 60.degree.
C. with 8 BU gds OPTIMALT.RTM. 2G with or without 0.5 AAU gds
SPEZYME.RTM. XTRA.
[0368] Each slurry was constantly shaken (orbital shaker incubator;
100 rpm) and samples were taken at different time intervals for
determination of sugar composition and concentration. The results
are shown in Table 5.
TABLE-US-00005 TABLE 5 Sugar compositions of high force milled corn
and corn liquefact treated with different alpha-amylases t DPn DP4
DP3 DP2 DP1 DP3 DP2 DP1 enzyme substrate [h] % % % % % (% w/V) (%
w/V) (% w/V) OPTIMALT .RTM. High 2 44.25 4.38 2.79 41.80 5.06 0.20
3.08 0.34 2G force 4 42.06 3.18 2.35 44.56 6.27 0.18 3.51 0.46 8 BU
milled 6 38.54 2.61 2.21 47.87 7.20 0.18 3.86 0.53 gds corn 20
30.25 1.63 1.70 55.83 9.02 0.14 4.71 0.70 25 30.45 1.68 1.08 56.55
8.77 0.09 4.94 0.71 30 28.57 1.45 1.58 57.65 9.25 0.13 4.97 0.73 45
27.41 1.38 1.59 58.44 9.71 0.13 5.02 0.77 50 27.05 1.36 1.54 58.68
9.91 0.13 5.06 0.78 Whole 2 31.48 4.30 4.78 50.23 8.48 0.34 3.60
0.56 ground 4 28.97 2.71 2.57 55.59 9.44 0.18 4.04 0.63 corn 6
27.51 1.99 1.44 58.50 9.87 0.12 4.77 0.74 liquefact 20 25.09 1.59
0.92 61.80 9.97 0.07 4.83 0.72 25 25.24 1.53 0.81 62.03 9.78 0.07
5.34 0.78 30 25.02 1.60 0.79 62.24 9.74 0.07 5.21 0.75 45 24.69
1.65 0.82 62.53 9.72 0.07 5.21 0.74 50 24.54 1.61 0.79 62.70 9.77
0.06 5.14 0.74 OPTIMALT .RTM. High 2 44.93 6.27 5.64 36.87 4.76
0.47 3.13 0.37 2G force 4 32.26 4.44 5.30 49.29 7.20 0.44 4.18 0.56
8 BU milled 6 27.75 2.98 3.44 55.54 8.79 0.29 4.70 0.68 gds + corn
20 23.42 1.83 1.50 61.08 10.67 0.13 5.24 0.84 SPEZYME .RTM. 25
21.52 1.61 1.61 63.51 10.35 0.15 6.02 0.90 XTRA 30 22.42 1.63 1.40
62.11 10.96 0.12 5.44 0.88 0.5 AAU 45 21.41 1.70 1.38 62.68 11.40
0.12 5.51 0.92 gds 50 22.89 1.80 1.43 61.20 11.25 0.13 5.57 0.94
Whole 2 28.17 4.22 5.27 52.39 9.22 0.39 3.90 0.63 ground 4 25.75
2.73 2.78 57.85 10.21 0.24 5.10 0.83 corn 6 24.49 2.03 1.56 60.43
10.79 0.13 5.08 0.83 liquefact 20 22.71 1.75 0.89 63.06 10.95 0.07
5.07 0.81 25 23.09 1.77 0.82 63.00 10.71 0.07 5.47 0.86 30 22.80
1.74 0.79 63.61 10.49 0.07 5.48 0.83 45 22.44 1.89 0.85 63.82 10.44
0.07 5.27 0.79 50 22.47 1.84 0.82 63.77 10.54 0.07 5.29 0.80
[0369] Table 5 shows that addition of OPTIMALT.RTM. 2G to high
force milled corn resulted in more than 58% DP2, which is mainly
maltose. With the reference liquefact a few percent more DP2 was
found, but in absolute numbers similar amounts of DP2 were
measured. The relative difference comes from a higher percentage of
DPn and DP3 for the high force milled corn, which again indicates
that high force milling results in a higher soluble substrate than
conventional liquefaction.
[0370] Addition of SPEZYME.RTM. XTRA resulted in lower DPn for both
samples, but the higher DP3, DP2, and DP1 concentrations (w/v %) in
the high force milled corn incubation may indicate that the
substrate was more accessible for OPTIMALT.RTM. 2G or that more
starch was solubilized by the high force milling. With the extra
alpha-amylase, higher percentages of DP2 were obtained for both
samples.
[0371] This example shows that high maltose syrups were produced
from corn without a liquefaction step by treating the corn with a
high force milling and incubating this material with a maltogenic
alpha-amylase with or without additional alpha-amylase. Also it
shows that high force milling resulted in a better substrate for
malto-saccharification than can be obtained via traditional
liquefaction.
Example 10
Maltose Production from High Force Milled Corn with a
Beta-Amylase
[0372] In this experiment a beta-amylase with pullulanase, with and
without additional alpha-amylase, was tested to see whether maltose
syrup can be made directly from high force milled corn without
application of liquefaction with alpha-amylase.
[0373] A 10% DS aqueous slurry of high force milled corn (prepared
as in Example 2) was incubated at pH 5.0 and 60.degree. C. with 4
DP gds OPTIMALT.RTM. BBA with 1.5 ASPU gds OPTIMAX.RTM. L-1000,
with or without 0.5 AAU gds SPEZYME.RTM. XTRA.
[0374] Each slurry was constantly shaken (orbital shaker incubator;
100 rpm) and samples were taken at different time intervals for
determination of sugar composition and concentration. The results
are shown in Table 6.
TABLE-US-00006 TABLE 6 Sugar compositions of high force milled corn
with different combinations of beta-amlyase, pullinase, and
alpha-amylase t DPn DP4 DP3 DP2 DP1 DP3 DP2 DP1 enzyme substrate
[h] % % % % % (% w/V) (% w/V) (% w/V) OPTIMALT .RTM. High 2 42.87
2.02 3.15 45.87 4.24 0.21 3.15 0.27 BBA force 4 44.34 2.60 3.87
42.48 4.96 0.27 3.00 0.32 4 DP milled 6 44.19 3.05 4.30 41.25 5.49
0.32 3.06 0.37 gds + corn 20 43.90 3.25 4.81 39.62 6.74 0.36 3.00
0.47 OPTIMAX .RTM. 25 44.15 3.47 5.36 38.84 6.60 0.42 3.10 0.49
L-1000 30 44.57 3.22 4.90 38.95 6.75 0.39 3.11 0.50 1.5 ASPU 45
42.53 3.58 5.32 39.68 7.23 0.39 2.96 0.50 gds OPTIMALT .RTM. High 2
34.75 4.19 6.20 50.07 3.26 0.52 4.22 0.25 BBA force 4 27.31 6.00
8.64 52.83 3.71 0.71 4.37 0.28 4 DP milled 6 23.80 6.91 10.00 53.87
3.90 0.81 4.43 0.30 gds + corn 20 19.34 7.95 11.71 55.19 4.28 0.96
4.60 0.33 OPTIMAX .RTM. 25 17.32 8.48 12.00 56.32 4.31 1.02 4.86
0.34 L-1000 30 18.17 8.36 11.85 55.76 4.31 0.99 4.71 0.34 1.5 ASPU
45 17.60 8.22 12.02 55.91 4.75 0.99 4.68 0.37 gds + SPEZYME .RTM.
XTRA 0.5 AAU gds
[0375] Table 6 shows that in a short time frame (<6 h) maltose
syrup was made from incubating high force milled corn with
beta-amylase and pullulanase.
[0376] Addition of alpha-amylase increased the percentage and
concentration of DP2, but also helped to hydrolyse the corn
further, as DP4% and DP3% were higher than without SPEZYME.RTM.
XTRA
[0377] This example shows that maltose syrups were produced from
corn without a liquefaction step by grinding the corn with a high
force mill and incubating this material with a beta-amylase and
pullulanase, with or without additional alpha-amylase.
Example 11
Maltotriose Production from High Force Milled Corn using an
Alpha-Amylase
[0378] In this experiment an alpha-amylase with and without
pullulanase was tested to see whether a syrup rich in DP3 can be
made directly from high force milled corn without application of
liquefaction with alpha-amylase. The results were compared to those
obtained with a traditional corn liquefact.
[0379] A 10% DS aqueous slurry of high force milled corn (prepared
as in Example 2) was incubated at pH 4.4 and 60.degree. C. with 3
SSU gds Aspergillus clavatus alpha-amylase with or without 1.081
ASPU gds OPTIMAX.RTM. L-1000.
[0380] As reference, corn liquefact (see General Methods) was
diluted to 10% DS and the slurry was incubated at pH 4.4 at
60.degree. C. with 3 SSU gds Aspergillus clavatus alpha-amylase
with or without 1.081 ASPU gds OPTIMAX.RTM. L-1000.
[0381] Each slurry was constantly shaken (orbital shaker incubator;
100 rpm) and samples were taken at different time intervals for
determination of sugar composition and concentration. The results
are shown in Table 7.
TABLE-US-00007 TABLE 7 Sugar compositions of high force milled corn
and corn liquefact treated with different combinations of
alpha-amylase and pullanase t DPn DP4 DP3 DP2 DP1 DP3 DP2 DP1
enzyme substrate [h] % % % % % (% w/V) (% w/V) (% w/V) AcAA High 2
57.69 11.32 18.42 8.47 2.54 1.55 0.72 0.20 3 SSU force 4 46.24
12.30 23.69 13.19 3.06 1.99 1.12 0.24 gds milled 6 40.88 11.84
26.00 16.32 3.38 2.19 1.39 0.27 corn 20 38.80 10.93 26.64 17.93
4.16 2.28 1.55 0.33 25 38.97 10.74 26.51 17.97 4.30 2.32 1.59 0.35
30 38.87 10.54 26.60 18.05 4.44 2.28 1.57 0.36 45 38.88 10.11 26.35
18.26 4.92 2.27 1.60 0.40 50 38.92 10.00 26.27 18.29 5.03 2.27 1.60
0.41 Whole 2 36.20 11.45 29.24 17.75 4.61 2.19 1.35 0.32 ground 4
33.96 10.05 30.14 20.55 4.60 2.35 1.62 0.33 corn 6 33.42 9.32 30.32
21.64 4.60 2.16 1.56 0.31 liquefact 20 33.06 8.89 30.29 22.36 4.73
2.22 1.66 0.32 25 33.70 8.79 30.05 22.12 4.67 2.43 1.81 0.35 30
33.78 8.69 29.99 22.21 4.69 2.27 1.70 0.33 45 33.93 8.40 29.82
22.36 4.83 2.26 1.72 0.34 50 33.87 8.52 29.79 22.34 4.82 2.22 1.68
0.33 AcAA High 2 52.92 12.94 20.75 9.25 2.61 1.76 0.80 0.21 3 SSU
force 4 33.65 16.31 29.49 15.66 3.28 2.34 1.26 0.24 gds + milled 6
29.36 15.76 31.66 18.28 3.39 2.62 1.53 0.26 OPTIMAX .RTM. corn 20
23.21 15.79 34.19 21.14 4.14 2.88 1.81 0.33 L-1000 25 23.38 15.64
33.95 21.22 4.31 2.98 1.89 0.35 1.081 ASPU 30 22.53 15.67 34.21
21.52 4.56 2.92 1.86 0.36 gds 45 22.90 15.17 33.71 21.55 5.17 2.88
1.87 0.41 50 22.78 14.91 33.84 21.74 5.25 2.93 1.91 0.42 Whole 2
29.24 15.08 32.48 17.91 4.58 2.55 1.42 0.34 ground 4 21.98 14.58
35.86 22.19 4.71 2.55 1.60 0.31 corn 6 17.90 14.84 37.23 24.41 4.93
2.84 1.89 0.35 liquefact 20 15.39 13.24 37.87 26.49 6.34 2.78 1.97
0.43 25 15.58 12.96 37.53 26.57 6.70 2.95 2.11 0.49 30 14.68 13.14
37.53 26.93 7.10 2.82 2.05 0.50 45 13.99 12.11 37.12 27.77 8.37
2.83 2.14 0.59 50 14.54 11.34 37.02 27.93 8.55 2.97 2.27 0.63
[0382] Table 7 shows that a DP3 rich syrup was produced from high
force milled corn by incubation with the Aspergillus clavatus
alpha-amylase (AcAA). A higher percentage and concentration of DP3
was obtained by addition of pullulanase. The concentrations of DP3
in the syrups produced from high force milled corn and whole ground
corn liquefact were similar, while the percentage of DP3 was lower
in the former compared to the latter. The percentages of DPn and
DP4 were higher for high force milled corn incubations than for the
reference reactions. DP1 was higher for high force milled corn,
than for the traditional liquefact.
[0383] Addition of OPTIMAX.RTM. L-1000 resulted for both substrates
in a decrease of DPn % and an increase in DP4%. DP2 content was
relatively and absolutely lower for high force milled corn
incubations, while DP4% and DPn % were higher. DP1 was lower for
the high force milled corn, than for the traditional liquefact.
This may indicate that the whole ground corn liquefact contains
limited dextrins, or similar structures, which result in more DP2
and DP1 upon hydrolysis by pullulanase, while the high force milled
corn consists of larger starch fragments. Hydrolysis by pullulanase
of these fragments resulted in more substrate for AcAA, so more DP3
was produced, but not more DP2 and DP1, as observed when comparing
results with and without OPTIMAX.RTM. L-1000.
[0384] This example shows that syrups rich in DP3 were produced
directly from corn without a liquefaction step by high force
milling the substrate and incubating with a DP3 producing
alpha-amylase, with or without a pullulanase. Also it was shown
that incubations of high force milled corn with a DP3 producing
alpha-amylase and pullulanase resulted in lower DP1 and DP2 than
incubations of liquefaction of corn with the same enzymes.
Example 12
Maltotetraose Production from High Force Milled Corn using an
Alpha-Amylase
[0385] In this experiment a DP4 producing alpha-amylase with and
without pullulanase was tested to see whether a syrup rich in DP4
can be made directly from high force milled corn without
application of liquefaction with alpha-amylase. The results were
compared to those obtained with a traditional corn liquefact.
[0386] A 10% DS aqueous slurry of high force milled corn (prepared
as described in Example 2) was incubated at pH 5.0 and 60.degree.
C. with 0.041 BMK gds OPTIMALT.RTM. 4G with or without 1.081 ASPU
gds OPTIMAX.RTM. L-1000.
[0387] As reference, corn liquefact (see General Methods) was
diluted to 10% DS and the slurry was incubated at pH 4.4 at
6.degree. C. with 0.041 BMK gds OPTIMALT.RTM. 4G with or without
1.081 ASPU gds OPTIMAX.RTM. L-1000.
[0388] Each slurry was constantly shaken (orbital shaker incubator;
100 rpm) and samples were taken at different time intervals for
determination of sugar composition and concentration (DP4% w/v was
estimated by extrapolating DP3's calibration curve). The results
are shown in Table 8.
TABLE-US-00008 TABLE 8 Sugar compositions of high force milled corn
and corn liquefact treated with DP4 producing alpha-amylase with
and without pullanase t DPn DP4 DP3 DP2 DP1 DP4 DP3 DP2 DP1 enzyme
substrate [h] % % % % % (% w/V) (% w/V) (% w/V) (% w/V) OPTIMALT
.RTM. High 2 55.18 34.21 4.62 1.23 2.98 2.60 0.35 0.09 0.21 4G
force 4 47.77 38.69 6.36 1.88 3.65 3.03 0.50 0.15 0.27 0.041 BMK
milled 6 41.87 41.36 8.16 2.52 4.50 3.36 0.66 0.21 0.34 gds corn 20
35.66 38.35 12.32 5.05 7.13 3.36 1.08 0.45 0.58 25 35.23 37.22
13.02 5.49 7.62 3.31 1.16 0.49 0.63 30 34.07 36.53 13.69 6.02 8.26
3.24 1.21 0.54 0.68 45 33.19 33.48 15.23 7.05 9.64 2.96 1.35 0.63
0.80 50 33.00 32.56 15.68 7.41 9.95 2.93 1.41 0.68 0.84 Whole 2
35.24 39.41 10.73 8.11 5.79 3.08 0.84 0.64 0.42 ground 4 33.74
40.43 10.75 8.48 5.93 3.00 0.80 0.64 0.41 corn 6 32.64 41.47 10.99
8.15 6.05 3.15 0.83 0.63 0.43 liquefact 20 30.49 40.09 12.98 8.62
7.20 3.16 1.02 0.69 0.53 25 30.80 39.19 13.43 8.63 7.37 3.18 1.09
0.71 0.56 30 30.25 38.65 13.94 8.83 7.74 3.16 1.14 0.73 0.59 45
29.73 36.20 15.46 9.42 8.63 3.13 1.34 0.82 0.70 50 29.82 35.53
15.77 9.44 8.89 3.12 1.38 0.84 0.73 OPTIMALT .RTM. High 2 48.32
39.50 5.53 1.67 3.25 3.14 0.44 0.13 0.24 4G force 4 35.74 47.92
7.90 2.72 4.10 3.81 0.63 0.22 0.30 0.041 BMK milled 6 25.99 53.58
10.29 3.53 5.08 4.39 0.84 0.29 0.39 gds + corn 20 11.33 56.80 15.92
6.54 7.95 4.97 1.39 0.58 0.65 OPTIMAX .RTM. 25 10.51 55.51 16.92
7.07 8.57 4.96 1.51 0.64 0.71 L-1000 30 8.61 54.77 18.16 7.68 9.34
4.83 1.60 0.69 0.77 1.081 ASPU 45 8.11 49.44 20.77 9.30 10.98 4.36
1.83 0.83 0.90 gds 50 8.88 47.93 20.98 9.42 11.39 4.26 1.86 0.85
0.94 Whole 2 24.97 46.82 12.46 8.81 6.23 3.42 0.91 0.65 0.42 ground
4 19.17 51.27 13.33 9.03 6.53 3.81 0.99 0.68 0.45 corn 6 13.48
54.91 14.49 9.48 6.98 4.37 1.15 0.76 0.52 liquefact 20 6.82 55.47
17.69 10.72 8.67 4.31 1.38 0.84 0.63 25 7.46 53.62 18.43 11.22 8.73
4.61 1.59 0.98 0.70 30 6.69 53.70 18.69 10.96 9.41 4.35 1.51 0.90
0.71 45 5.50 50.06 21.20 11.81 10.92 4.32 1.83 1.03 0.88 50 5.34
49.05 21.73 12.08 11.27 3.95 1.75 0.98 0.85
[0389] Table 8 shows that incubations with OPTIMALT.RTM. 4G with
and without OPTIMAX.RTM. L-1000 resulted in similar DP4 percentages
for the high force milled corn as for the whole ground corn
liquefact.
[0390] With and without OPTIMAX.RTM. L-1000, the incubations with
high force milled corn resulted in higher DP1, lower DP2 and again
higher DP3 concentrations than incubations with corn liquefact.
This indicates that the soluble substrate obtained by high force
milling has a different structure than the material obtained by
liquefaction of whole ground corn.
[0391] Without OPTIMAX.RTM. L-1000, DP4 was slightly lower for high
force milled corn than for corn liquefact, but with OPTIMAX.RTM.
L-1000 the experiment with high force milled corn resulted in a
higher concentration of DP4 than with liquefact. This means that
high force milling provided a better substrate than
liquefaction.
[0392] This example shows that syrups rich in DP4 were produced
directly from corn without a liquefaction step by high force
milling the substrate and incubating with a DP4 producing
alpha-amylase with or without a pullulanase. It also shows that the
yield obtained when OPTIMALT.RTM. 4G and OPTIMAX.RTM. L-1000 were
incubated with high force mill treated corn, was higher than when
incubated with liquefact.
Example 13
Maltopentaose Production from High Force Milled Corn using an
Alpha-Amylase
[0393] In this experiment a DP5 producing alpha-amylase with and
without pullulanase was tested to see whether a syrup rich in DP5
can be made directly from high force milled corn without
application of liquefaction with alpha-amylase. The results were
compared to those obtained with a traditional corn liquefact.
[0394] A 10% DS aqueous slurry of high force milled corn (prepared
as described in Example 2) was incubated at pH 5.0 and 60.degree.
C. with 3 .mu.g gds Cytophaga sp. DP5 producing alpha-amylase
(DP5AA) with or without 1.081 ASPU gds OPTIMAX.RTM. L-1000.
[0395] As reference corn liquefact (see General Methods) was
diluted to 10% DS and the slurry was incubated at pH 4.4 at
60.degree. C. with 3 .mu.g gds DP5 producing alpha-amylase with or
without 1.081 ASPU gds OPTIMAX.RTM. L-1000.
[0396] Each slurry was constantly shaken (orbital shaker incubator;
100 rpm) and samples were taken at different time intervals for
determination of sugar composition and concentration. The results
are shown in Table 9.
TABLE-US-00009 TABLE 9 Sugar compositions of high force milled corn
and corn liquefact treated with a DP5 producing alpha-amylase with
and without pullulanase t DPn DP10 DP9 DP8 DP7 DP6 DP5 DP4 DP3 DP2
DP1 enzyme substrate [h] % % % % % % % % % % % DP5 AA High 2 34.35
1.18 1.46 1.99 7.73 13.74 12.41 4.61 10.60 6.68 5.24 3 .mu.g force
4 32.20 1.19 1.33 1.54 6.16 13.12 13.94 5.23 11.81 8.05 5.40 gds
milled 20 23.63 1.85 1.99 1.96 2.49 12.04 21.21 5.43 13.32 9.32
6.76 corn 25 23.19 1.73 1.98 1.94 2.45 11.72 21.50 5.61 13.46 9.47
6.92 30 23.05 1.73 2.02 1.96 2.40 11.44 21.65 5.73 13.52 9.52 7.00
45 22.92 1.71 2.05 2.00 2.39 10.94 21.48 6.01 13.60 9.61 7.29 50
23.04 1.65 2.05 2.01 2.38 10.83 21.36 6.13 13.55 9.63 7.37 Whole 2
55.05 1.63 2.11 3.05 7.25 7.81 7.76 2.73 7.07 2.94 2.61 ground 4
39.68 1.40 1.67 2.25 6.93 10.81 12.93 4.20 10.74 5.35 4.03 corn 20
21.83 1.95 2.16 2.11 2.40 8.65 20.57 7.23 13.33 10.73 9.04
liquefact 25 20.97 1.90 2.11 2.06 2.33 8.17 20.46 7.55 13.55 11.24
9.67 30 20.40 1.87 2.10 2.06 2.30 7.72 20.26 7.79 13.73 11.65 10.11
45 19.84 1.72 2.04 2.04 2.27 6.92 19.19 8.28 14.20 12.58 10.93 50
19.97 1.65 2.03 2.02 2.29 6.76 18.83 8.37 14.28 12.75 11.05 DP5 AA
High 2 28.01 1.56 2.33 3.44 9.97 14.47 11.91 5.52 11.40 6.39 5.02 3
.mu.g force 4 19.79 1.21 1.86 2.82 10.13 16.22 14.77 6.35 14.01
7.42 5.41 gds + milled 20 4.45 0.35 0.73 1.51 4.42 17.69 25.43 8.35
18.85 11.32 6.89 OPTIMAX .RTM. corn 25 3.81 0.32 0.60 1.38 3.83
17.04 26.03 8.61 19.40 11.80 7.19 L-1000 30 3.64 0.30 0.49 1.24
3.49 17.19 26.82 8.53 19.28 11.85 7.18 1.081 ASPU 45 3.67 0.12 0.41
1.06 3.00 16.77 27.24 8.75 19.45 12.11 7.43 gds 50 3.53 0.33 0.39
1.05 2.90 16.66 27.18 8.84 19.45 12.21 7.46 Whole 2 42.74 2.47 3.27
4.68 9.47 9.35 9.08 3.68 9.03 3.38 2.85 ground 4 27.14 1.25 1.80
2.96 10.60 13.02 13.79 5.57 13.98 5.75 4.14 corn 20 4.58 0.39 0.60
1.15 2.58 13.31 25.88 10.10 19.45 13.01 8.95 liquefact 25 4.39 0.33
0.48 1.02 2.24 12.54 25.96 10.37 19.73 13.57 9.38 30 3.48 0.30 0.42
0.95 2.03 11.99 25.97 10.72 20.13 14.16 9.83 45 3.79 0.43 0.35 0.81
1.72 10.59 24.83 11.21 20.56 15.13 10.57 50 3.95 0.33 0.35 0.78
1.66 10.34 24.45 11.35 20.69 15.37 10.74
[0397] Table 9 shows that from high force milled corn a DP5 rich
syrup was produced by incubating the substrate with DP5 AA. It can
be seen that a higher percentage of DP5 and DP6 was produced with
high force milled corn in comparison with whole ground corn
liquefact. The incubation with the latter resulted in higher DP1,
DP2, DP3, and DP4. This indicates that the substrate available for
hydrolysis had a larger polymer size than that found in liquefied
corn, since the larger percentage of smaller sugars indicates more
undesirable sugars (sugars other than maltopentaose) from
hydrolysis. This resulted in a higher percentage of the desired
maltodextrins; in this case DP5.
[0398] The results showed that addition of OPTIMAX.RTM. L-1000
increased the percentage of DP5 by hydrolysing DPn as well as
reducing DP8-DP10. The differences between substrates can again be
seen with addition of OPTIMAX.RTM. L-1000; incubation with high
force milled corn resulted in lower DPn than with liquefact, DP6
and DP5 were higher, while DP1-DP4 were lower.
[0399] This example shows that syrups rich in DP5 were produced
directly from corn without a liquefaction step by high force
milling the substrate and incubating with a DP5 producing
alpha-amylase with or without a pullulanase. It showed that corn
treated by a high force mill resulted in a better substrate for
this application than corn traditionally liquefied.
Example 14
Production of Fermentable Sugars During Hydrolysis of High Force
Milled Corn
[0400] An experiment was performed to compare high force milled
corn (prepared as described in Example 2) and corn flour in
production of glucose during direct corn to fermentable sugar
production at pH 4.5 and 60.degree. C. The experiments were carried
out using 20% dry solids, with DI water making up the 100% of the
liquid phase of a slurry of each sample. The pH of each slurry was
adjusted to pH 4.5. A 35 g aliquot of each corn slurry with 2
replications for each treatment was weighed into LABOMAT steel
beakers. Following the weighing process the enzymes as given below
in Table 10 were dispersed in each slurry. The metal beakers were
fitted back into the LABOMAT for incubation at 60.degree. C. for 48
hrs with clockwise and counter-clockwise constant mixing at 60 rpm.
Samples were drawn at16, 24 and 48 hrs to analyze the percent
solubility and saccharide profile (shown in Table 10).
[0401] The alpha-amylase used was SPEZYME.RTM. RSL (a blend of
alpha-amylase and phytase), which was added at 8 AAUs/gds and the
glucoamylase used was AfuGA, which was added at 0.2 GAUs/gds.
Percent solubilization and saccharide profiles at various intervals
are given in Table 10.
TABLE-US-00010 TABLE 10 Percent solubilization and saccharide
profiles for high force milled corn and corn flour treated with
alpha-amylase and glucoamylase % % % % % g glucose/ sample enzyme
sol. hr HS DP3 DP2 DP1 liter high 8 AAUs 97.5 16 3.95 1.46 4.09
90.49 152.1 force 0.2 98.1 24 2.40 1.23 4.09 92.28 156.2 milled
GAUs 98.4 48 2.78 0.85 4.25 92.12 156.4 corn Corn 8 AAUs 90.7 16
6.01 0.95 5.57 87.46 136.8 flour 0.2 92.2 24 4.75 0.77 5.20 89.27
142.0 GAUs 92.9 48 4.51 0.43 4.30 90.76 145.4
[0402] The DP1 production with increasing percent solubilization
shown in Table 10 is evidence of ring and puck mill ground corn
yielding higher solubility and providing a higher amount of glucose
throughout the incubation process as compared to the ground
corn.
Example 15
Use of High Force Milled Corn in a No-Cook Simultaneous
Saccharification and Fermentation Process
[0403] High force milled (ring and puck milled) corn was evaluated
for performance in a fuel ethanol production process using a
no-cook simultaneous saccharification and fermentation (SSF)
process. In this example high force milled corn (prepared as
described in Example 2) was subjected to different gluco-amylase
(GA) enzyme dosages and compared to a coarse whole ground corn
(39%<500 .mu.m) and a corn flour substrate (85%<500 .mu.m).
The experiments were carried out using a 22% dry solids corn
slurry, solid urea was added to a final concentration of 800 ppm
and the pH was adjusted to 4.5 using 6 N sulfuric acid. 50 grams
(+/-0.2 g) of each slurry was weighed into replicate 125 ml
Erlenmeyer flasks and 6 SSU/g ds Aspergillus kawachii alpha-amylase
(AkAA) and 0.1 SAPU/g ds FERMGEN.TM. 2.5x was added to each flask.
0.1% w/w of FERMAX.TM. GOLD yeast (Martrex, Inc. Minnetonka, Minn.)
was added to each flask as well. Trichoderma reesei GA (TrGA) was
added to the flasks in the following dosages: 0.325-0.5-1.0 GAU/g
ds. The control in this experiment was the fine milled corn flour
with a TrGA dosage of 1.0 GAU/g ds and an AKAA dosage of 6 SSU/g
ds. All flasks were incubated at 32.degree. C. for up to 90 hours.
Time point samples were collected throughout the saccharification
and fermentation (SSF) at t=0, 4, 16, 30, 44, 55 and 72 hours.
[0404] Following SSF, each time point sample was prepared and
analyzed via HPLC for ethanol, DP1, DP2, DP3, DP4+, and glycerol.
Each sample was allowed to thaw at 4.degree. C. and was centrifuged
at 15,000 rpm for 5 minutes. 100 .mu.l of each sample supernatant
was incubated with 10 .mu.l of 1.1 N sulfuric acid for 5 minutes at
100.degree. C. Following incubation, 1 ml of Milli-Q water was
added to each sample and all samples were filtered with a 0.22
.mu.m filter. 20 .mu.l of each sample was injected into an Agilent
1200 series HPLC and run on a Phenomenex Rezex-RFQ Fast Fruit
column (cat #00D-0223-K0) with a Phenomenex Rezex ROA Organic Acid
guard column (cat #03B-0138-K0), heated to 85.degree. C., using a 9
minute isocractic elution in 0.01 N sulfuric acid at 1 ml/min.
Component concentrations were determined from a five point
calibration curve using a Supelco Fuel Ethanol standard (Sigma cat
#48468-U).
[0405] Table 11 shows the yields for ethanol and glycerol during up
to 44 hrs of SSF for the different substrates. The initial rate of
ethanol production (over the first 15 min) was much higher for the
ring and puck milled corn when compared to the coarse ground whole
corn and the fine milled corn flour for all of the enzyme dosages
tested. After 44 hrs no further increase in ethanol yield was
observed. Maximum ethanol yield for the ring and puck milled corn
was obtained within 44 hrs and was obtained with a 3.times. lower
enzyme dosage (0.325 GAU/g ds) as compared to the control (corn
flour treated with 1.0 GAU/g ds enzyme dosage. Coarse ground whole
corn was not effectively hydrolyzed using the no-cook process. More
glycerol was produced when more ethanol was produced.
TABLE-US-00011 TABLE 11 SSF yields during first 44 hr of
fermentation for ethanol and glycerol for high force milled corn,
corn flour and coarse whole ground corn EtOH conc (v/v %) Glycerol
conc (w/v %) TrGA Ring and Ring and dosage Puck Whole Puck Whole
(GAU/ Milled Corn Ground Milled Corn Ground g ds) Time Corn Flour
Corn Corn Flour Corn 0.325 0 0 0 0 0.04 0.03 0.04 4 0.53 0.61 0.59
0.15 0.11 0.12 16 8.57 3.63 1.61 0.47 0.15 0.11 30 10.18 4.99 1.98
0.44 0.09 0.05 44 9.45 6.18 2.38 0.47 0.04 0.03 0.5 0 0 0 0 0.04
0.03 0.04 4 0.52 0.61 0.57 0.15 0.12 0.11 16 8.83 4.07 1.73 0.52
0.17 0.11 30 10.12 5.75 2.31 0.50 0.10 0.06 44 9.47 6.85 2.67 0.53
0.05 0.03 1.0 0 0 0 0 0.04 0.03 0.04 4 0.49 0.61 0.71 0.15 0.13
0.13 16 10.08 5.03 2.53 0.64 0.20 0.13 30 10.02 6.89 3.57 0.63 0.13
0.08 44 9.50 8.08 3.96 0.67 0.07 0.04
Example 16
Enzyme Dose Reduction for High Force Milled Corn in a No-Cook
Simultaneous Saccharification and Fermentation Process (No-Cook SSF
Process)
[0406] High force milled (ring and puck milled) corn was further
evaluated after the initial observation that a 3.times. enzyme dose
reduction as compared to the corn flour control was effective in
the no-cook SSF process. A similar experimental set-up was used as
described in Example 13 with the following deviation: Trichoderma
reesei GA (TrGA) was added to the flasks in the following dosages
for the ring and puck milled corn: 0.1-0.2-0.3 GAU/g ds; and for
the corn flour control: 0.5 GAU/g ds and 1.0 GAU/g ds.
[0407] Table 12 shows the yields for ethanol for up to 60 hrs of
fermentation for the two substrates and the different enzyme
dosages applied. Initial ethanol formation rate was faster for ring
and puck milled corn for all enzyme dosages tested compared to the
corn flour control. The standard enzyme dosage in the no-cook
process is 1.0 GAU/g ds and the data show that a 10.times. reduced
dose (0.1 GAU/g ds) was successful when using the ring and puck
milled corn, which has not been observed with any other milled
sample. In addition, maximal amounts of ethanol were produced in
less time for the ring and puck milled corn.
[0408] Table 13 shows the residual starch after 72 hrs of
fermentation for the ring and puck milled corn and corn flour at
the different enzyme dosages applied. Residual starch data clearly
show more complete hydrolysis of the ring and puck milled corn as
compared to the corn flour and emphasizes the observation that this
was obtained with a 10.times. lower enzyme dose when using ring and
puck milled corn.
TABLE-US-00012 TABLE 12 SSF yields during first 60 hrs of
fermentation for ethanol for 5 min ring and puck milled corn and
corn flour at different enzyme dosages EtOH conc (v/v %) Ring and
Puck Milled Corn Corn Flour 0.1 0.2 0.3 0.5 1.0 Time GAU/g ds GAU/g
ds GAU/g ds GAU/g ds GAU/g ds 0 0 0 0 0.09 0.09 4 0.46 0.42 0.42
0.53 0.53 16 8.38 10.37 9.93 5.29 5.88 38 12.33 12.66 12.59 8.18
9.55 44 12.37 12.54 12.45 8.63 10.20 60 11.62 11.48 11.33 9.76
10.73
TABLE-US-00013 TABLE 13 Residual starch (% DW) after 72 hrs for
puck mlled corn and corn flour at different enzyme dosages Residual
starch (% DW) Ring andPuck Milled Corn Corn Flour 0.1 GAU/g ds 0.2
GAU/g ds 0.3 GAU/g ds 0.5 GAU/g ds 1.0 GAU/g ds 6.14 5.36 5.08
45.15 22.23
Example 17
Effect of Milling Time in the Ring and Puck Mill on Required Enzyme
Dosage in the No-Cook Simultaneous Saccharification and
Fermentation Process (No-Cook SSF Process)
[0409] Corn kernels were treated in the ring and puck mill as
described in example 2, except that the mill was run for 30 sec, 1
min and 5 min for different samples. These samples were evaluated
in the no-cook SSF process to assess the enzyme dosage needed to
convert the samples that were treated for different times. A
similar experimental set-up was used as described in Example 13
with the following deviations: (1) 20% dry solids 30 sec, 1 min,
and 5 min ring and puck milled corn substrates were used, (2)
Trichoderma reesei GA (TrGA) was added to the flasks in the
following dosages for the 30 sec, 1 min and 5 min ring and puck
milled corn samples: 0.1-0.3-0.5 GAU/g ds, and for the corn flour
control: 0.5 GAU/g ds and 1.0 GAU/g ds.
[0410] Table 14 shows the yields for ethanol up to 67 hrs of
fermentation for the 30 sec, 1 min and 5 min ring and puck milled
corn samples using different enzyme dosages; corn flour was used as
the control. 30 sec ring and puck milled corn when dosed with
0.1-0.3 GAU/g ds of gluco-amylase did not reach the same final
ethanol concentration as the 5 min ring and puck milled corn sample
reached after 63 hrs. and clearly more enzyme is needed to finish
the fermentation. Increasing the milling time from 30 sec to 1 min
results in a faster fermentation with an enzyme dosage of 0.3 GAU/g
ds of gluco-amylase for the 1 min ring and puck milled corn
compared to the 30 sec ring and puck milled corn. Increasing the
milling time from 1 min to 5 min shows that it is possible to
obtain highest ethanol with only 0.1 GAU/g ds of gluco-amylase for
the 5 min ring and puck milled corn whereas at least 0.3 GAU/g ds
is needed for 1 min ring and puck milled corn. Clearly the time of
milling can influence the needed enzyme dosage for an efficient
fermentation.
[0411] Table 15 shows the residual starch after 97 hrs of
fermentation for the 5 min ring and puck milled corn and 1 min ring
and puck milled corn at the different enzyme dosages applied.
Residual starch data clearly show the more complete hydrolysis of
the 1 min and 5 min ring and puck milled corn compared to the corn
flour and emphasizes the observation that this can be obtained with
a 10.times. lower enzyme dose when using a 5 min ring and puck
milled corn and a 3.times. lower enzyme dose when using 1 min ring
and puck milled corn.
[0412] Table 16 shows the amount of free oil that was detected at
the end of the fermentation. More free oil could be obtained from
ring and puck milled corn than the control corn flour. This
indicates that the high force milling has the potential to free up
more oil from the corn than traditional milling.
TABLE-US-00014 TABLE 14 SSF yields during first 67 hrs of
fermentation for ethanol for 30 sec, 1 min, and 5 min ring and puck
milled corn and corn flour at different enzyme dosages EtOH conc
(v/v %) Enzyme 30 sec Ring and 1 min Ring and 5 min Ring and Time
dosage Puck Milled Corn Puck Milled Corn Puck Milled Corn Corn
Flour (hrs) (GAU/g ds) 0.1 0.3 0.3 0.5 0.1 0.3 0.5 1.0 0 0.07 0.07
0.10 0.10 0.07 0.07 0.05 0.05 7 1.79 1.67 2.03 1.87 1.31 1.54 1.68
1.78 14 5.63 6.43 --* -- 4.93 7.46 3.53 3.75 17 -- -- 7.64 8.35 --
-- -- -- 30 7.10 7.74 8.73 9.19 8.72 10.21 4.80 5.44 44 7.30 8.33
9.07 9.39 9.70 9.75 5.97 6.63 63 7.51 8.39 -- -- 9.63 9.27 6.27
6.35 67 -- -- 8.31 8.51 -- -- -- -- *sample was not taken
TABLE-US-00015 TABLE 15 Residual starch (% DW) after 97 hrs for 5
min ring and puck milled corn and 1 min ring and puck milled corn
at different enzyme dosages. Corn flour is the control. Residual
starch (% DW) 1 Min Ring and Puck 5 Min Ring and Milled Corn Puck
Milled Corn Corn Flour 0.3 0.5 0.1 0.5 1.0 GAU/g ds GAU/g ds GAU/g
ds GAU/g ds GAU/g ds 5.48 4.10 3.44 17.89 12.33
TABLE-US-00016 TABLE 16 Detected free oil after 97 hours of
fermentation for 30 sec, 1 min, 5 min ring and puck milled corn and
corn flour Sample .mu.g free oil Corn Flour/1.0 GAU Below detection
limit 30 sec ring and puck milled corn, 0.3 GAU 170 1 min ring and
puck milled corn, 0.3 GAU 226 5 min ring and puck milled corn, 0.3
GAU 205
Example 18
Use of High Force Milled Starch Substrates in a No-Cook
Simultaneous Saccharification and Fermentation (SSF) Process
[0413] High force milled (puck milled) cassava, rice, sorghum and
wheat were evaluated for performance in a fuel ethanol production
process using a no-cook simultaneous saccharification and
fermentation (SSF) process. In this example high force milled
starch containing substrates (prepared as described in Example 1)
were compared to a conventionally milled substrate (equivalent to
corn flour substrate (85%<500 .mu.m)). The experiments were
carried out using 20% dry solids slurry, solid urea was added to a
final concentration of 800 ppm and the pH was adjusted to 4.5 using
6N sulfuric acid. 50 grams (+/-0.2 g) of each slurry was weighed
into replicate 125 ml Erlenmeyer flasks and 6 SSU/g ds Aspergillus
kawachii alpha-amylase (AkAA) and 0.1 SAPU/g ds FERMGEN.TM. 2.5x
were added to each flask. A 0.1% w/w of FERMAX.TM. GOLD yeast
(Martrex, Inc. Minnetonka, Minn.) was added to each flask as well.
Trichoderma reesei GA (TrGA) was added to the flasks at the 0.3
GAUs/g ds for the high force milled substrate. The control in this
experiment was the conventionally milled flour for each of the
starch containing substrate with a TrGA dosage of 1.0 GAU/g ds and
an AKAA dosage of 6 SSU/g ds. All flasks were incubated at
32.degree. C. for up to 72 hours. Time point samples were collected
throughout the saccharification and fermentation (SSF) at t=0, 11,
15, 23, 30, 45, 55 and 72 hours.
[0414] Following SSF, each time point sample was prepared and
analyzed via HPLC for ethanol, DP1, DP2, DP3, DP4+, lactic acid,
acetic acid and glycerol. Each sample was allowed to thaw at
4.degree. C. and was centrifuged at 15,000 rpm for 5 minutes. 100
.mu.l of each sample supernatant was incubated with 10 .mu.l of 1.1
N sulfuric acid for 5 minutes at 100.degree. C. Following
incubation, 1 ml of Milli-Q water was added to each sample and all
samples were filtered with a 0.22 .mu.m filter. 20 .mu.l of each
sample was injected into an Agilent 1200 series HPLC and run on a
Phenomenex Rezex-RFQ Fast Fruit column (cat #00D-0223-K0) with a
Phenomenex Rezex ROA Organic Acid guard column (cat #03B-0138-K0),
heated to 85.degree. C., using a 9 minute isocractic elution in
0.01 N sulfuric acid at 1 ml/min. Component concentrations were
determined from a five point calibration curve using a Supelco Fuel
Ethanol standard (Sigma cat #48468-U).
[0415] Table 17 shows the yields for ethanol during up to 45 hrs of
SSF for the different substrates. The initial rate of ethanol
production (over the first 15 hr) was much higher for the puck
milled substrate (except for rice) when compared to the
conventionally milled substrate for the enzyme dosages tested.
After 45 hrs no further increase in ethanol yield was observed.
Maximum ethanol yield for the puck milled corn was obtained within
45 hrs and was obtained with a 3.times. lower enzyme dosage (0.3
GAU/g ds) as compared to the control (conventionally milled
substrate treated with 1.0 GAU/g ds enzyme dosage.
TABLE-US-00017 TABLE 17 Cold cook SSF yields for various starch
containing substrates during first 45 hr of fermentation for
ethanol (% v/v) Conventional Conventional Conventional Conventional
Puck milled milled Puck Milled milled Puck Milled milled Puck
Milled milled Cassava Cassava Rice Rice Sorghum Sorghum Wheat Wheat
Hrs 0.3 GAUs/g ds 1 GAUs/g ds 0.3 GAUs/g ds 1 GAUs/g ds 0.3 GAUs/g
ds 1 GAUs/g ds 0.3 GAUs/g ds 1 GAUs/g ds 11 2.39 2.04 1.46 1.92
1.71 2.44 2.07 2.02 15 5.52 2.85 3.70 4.95 4.45 3.67 5.04 2.47 23
7.47 3.78 6.30 6.19 6.54 4.75 6.74 3.03 30 7.98 4.58 7.67 6.80 7.53
5.35 7.03 3.49 45 8.21 5.58 8.36 7.52 7.67 6.09 7.05 4.05
* * * * *