I 1111111111111111 11111 111111111111111lllll 111111111111111 111111111111111111

US007968318B2

c12) United States Patent (IO) PatentNo.: US 7,968,318 B2

Lantero et al. (45) Date of Patent: Jun. 28, 2011

(54) PROCESS FOR CONVERSION OF FOREIGN PATENT DOCUMENTS

GRANULAR STARCH TO ETHANOL EP 0 171 218 A2 2/1986
WO WO 84/02921 A2 8/1984
(75) Inventors: Oreste J. Lantero, Beloit, WI (US); WO WO 92/00381 Al WO 1/1992
WO 95/13362 Al WO 5/1995
Mian Li, Beloit, WI (US); Jayarama K. WO 96/23874 Al WO 8/1996
Shetty, Palo Alto, CA (US) WO 96/39528 A2 WO 12/1996
WO 97/38096 Al WO 10/1997
WO
(73) Assignee: Genencor International, Inc., Palo 97/41213 Al WO 11/1997
WO 98/28408 Al WO 7/1998
Alto, CA (US) WO 98/28409 Al WO 7/1998
WO 98/44125 Al WO 10/1998
( *) Notice: Subject to any disclaimer, the term ofthis WO 99/19467 Al WO 4/1999
WO 99/28488 A2 WO
patent is extended or adjusted under 35 6/1999
WO 00/04136 Al WO 1/2000
U.S.C. 154(b) by 1185 days. WO 03/018766 A3 WO 3/2003
WO 03/066826 A2 WO 8/2003
WO 03/068976 A2 WO
(21) Appl. No.: 11/447,554 8/2003
WO 2004/080923 A2 WO 9/2004
WO 2004/081193 A2 9/2004
(22) Filed: Jun.6,2006 WO WO 2004/106533 Al 12/2004
WO WO 2004/111218 A2 12/2004
WO WO 2005/001064 A2 1/2005
(65) Prior Publication Data WO WO 2005/003311 A2 1/2005
US 2007/0281344 Al Dec. 6, 2007 WO WO 2005/045018 Al 5/2005
WO WO 2005/052148 A2 6/2005
WO WO 2005/069840 A2 8/2005
(51) Int. Cl. WO WO 2005/096804 A2 10/2005
C12P 19114 (2006.01) WO WO 2005/117756 A2 12/2005
WO WO 2005/118800 Al 12/2005
C12P 7106 (2006.01) WO WO 2006/043178 A2 4/2006
C12Q 1102 (2006.01)
OTHER PUBLICATIONS
C12Q 1148 (2006.01)
Cl2Q 1/34 (2006.01) Perpete et al., "Influence of Beer Ethanol Content on the Wort Flavour
Cl2Q 1/37 (2006.01) Perception", Food Chemistry, 71 (2000) 379-385.*
(52) U.S. Cl...................435/99; 435/161; 435/29; 435/23; Wikipedia, http://en.wikipedia.org/wiki/Starch_gelatinization pp.
1-3, printed Nov. 6, 2010.*
435/18; 435/15
U.S. Appl. No. 11/102,188, filed Jan. 19, 2006, Ferrari, et al.
(58) Field of Classification Search........................435/15, Allison, D.S. et al. "Transformation of the thermophilic fungus
435/18, 23, 29, 99, 161 Humicola grisea var. thermoidea and overproduction of Humicola
See application file for complete search history. glucoamylase," Current Genetics 21(3):225-229, Mar. 1, 1992.
Ashikari, T. et al. "Rhizopus raw-starch-degrading glucoamylase:its
cloning and expression in yeast," Agricultural and biological chem
(56) References Cited istry 50(4):957-964, 1986.
Berka, R.M. et al. "Molecular Characterization and Expression of a
U.S. PATENT DOCUMENTS Phytase Gene from the Thermophilic Fungus Thermomyces
4,092,434 A 5/1978 Yoshizumi et al. lanuginosus,"Appl. Environ. Microbial. 64(11):4423-4427, Nov. 1,
4,316,956 A 2/1982 Lutzen 1998.
4,514,496 A 4/1985 Yoshizumi et al. Boe!, E. et al. "Glucoamylases G1 and G2 fromAspergillus niger are
RE32,153 E 5/1986 Tamura et al. synthesized from two different but closely related mRNAs," The
4,587,215 A 5/1986 Hirsh EMBO Journal 3(5):1097-1102, May 1984.
4,618,579 A 10/1986 Dwiggins et al.
4,863,864 A 9/1989 Ashikari et al. (Continued)
5,000,000 A 3/1991 Ingram et al.
5,028,539 A 7/1991 Ingram et al. Primary Examiner - Jon P Weber
5,093,257 A 3/1992 Gray
5,424,202 A 6/1995 Ingram et al. Assistant Examiner - Kailash C Srivastava
5,514,583 A 5/1996 Picataggio et al. (74) Attorney, Agent, or Firm - Drinker Biddle & Reath
5,554,520 A 9/1996 Fowler et al. LLP
5,763,385 A 6/1998 Bott et al.
5,824,532 A 10/1998 Barnett et al.
5,863,533 A 1/1999 Van Gorcom et al.
(57) ABSTRACT
5,958,739 A 9/1999 Mitchinson et al. A method is disclosed for producing glucose from a
6,008,026 A 12/1999 Day granular starch substrate including, contacting a slurry
6,093,562 A 7/2000 Bisgard-Frantzen et al.
6,187,576 Bl 2/2001 Svendsen et al. comprising granular starch obtained from plant material
6,350,602 Bl 2/2002 Van Gorcom et al. with an alpha amylase at a temperature below the starch
6,352,851 Bl 3/2002 Nielsen et al. gelatinization tem perature of the granular starch to produce
6,361,809 Bl 3/2002 Christophersen et al. oligosaccharides and hydrolyzing the oligosaccharides to
6,734,004 B2 5/2004 Kostrewa et al. produce a mash com prising at least 20% glucose and
6,867,031 B2 3/2005 Bisgard-Frantzen et al.
7,413,887 B2 8/2008 Dunn-Coleman et al. further comprising ferment ing the mash to obtain ethanol.
2008/0318284 Al * 12/2008 Soong et al.........................435/96
2009/0098624 Al * 4/2009 Deinhammer et al..........435/161 28 Claims, 1 Drawing Sheet
 US 7,968,318 B2
Page 2

OTHER PUBLICATIONS
Shibuya, I. et al. "Overproduction of an a-Amylase/Glucoarnylase
Bothast, R.J. et al. "Biotechnological processes for conversion of
corn into ethanol," Applied Microbiology and Biotechnology Fusion Protein in Aspergillus oryzae Using a High Expression Vec
67(1):19-25, Apr. 17, 2005. tor," Bioscience, biotechnology, and biochemistry 56(10):1674-
Chen, H.M. et al. "Substitution ofasparagine residues inAspergillus 1675, 1992.
awamori glucoarnylase by site-directed mutagenesis to eliminate N- Suzuki, Y et al. "Amino acid residues stabilizing a Bacillus alpha
glycosylation and inactivation by dearnidation," Biochem. J. 30l(Pt amylase against irreversible thermoinactivation," J Biol. Chem.
1):275-281, Jul. 1, 1994. 264(32):18933-18938, Nov. 15, 1989.
Chen, H.-M. et al. "Identification and elimination by site-directed Swinkels, J.J.M. "Sources of Starch:Its Chemistry and Physics," In
mutagenesis ofthermolabile aspartyl bonds inAspergillus awamori Starch Conversion Technology, edited by G.M.A. van Beynum et
glucoarnylase," Protein Eng. 8(6):575-582, Jun. 1, 1995. al., pp. 32-38. NewYork:M. Dekker, 1985.
Chen, H.-M. et al. "Effect of replacing helical glycine residues with Takahashi, T. et al. "Different Behavior towards Raw Starch of
alanines on reversible and irreversible stability and production of
Three Forms ofGlucoarnylase from aRhizopus Sp," J Biochem
Aspergillus awamori glucoarnylase," Protein Eng. 9(6):499-505,
98(3):663- 671, Sep. 1, 1985.
Jun. 1, 1996.
Taylor, P.M. et al. "Some Properties of a Glucoarnylase Produced
Cornett, C.A.G. et al. "Starch-binding domain shuffling in Aspergil
lus niger glucoarnylase," Protein Eng. 16(7):521-529, Jul. 1, 2003. by the Thermophilic Fungus Humicola lanuginosa," Carbohydrate
Greiner, R. et al. "Purification and characterization of two phytases Research 61:301-308, 1978.
fromEscherichia coli,"Arch. Biochem. Biophys 303(1):107-13, May Tosi, L.R.O. et al. "Purification and characterization of an extracel
15, 1993. lular glucoarnylase from the thermophilic fungus Humicola grisea
Hata, Y. et al. "The glucoarnylase cDNA fromAspergillus oryzae:its var. thermoidea," Canadian Journal of Microbiology 39(9):846-852,
cloning, nucleotide sequence, and expression in Saccharomyces 1993.
cerevisiae," Agric. Biol. Chem. 55(4):941-949, Apr. 1991. Wodzinski, R.J. et al. "Phytase," In Advances in Applied Microbiol
Hayashida, S. et al. "Molecular cloning of the glucoarnylase I gene ogy, edited by S.L. Neidleman et al., vol. 42:pp. 263-302. New
of Aspergillus awamori var. kawachi for localization of the raw- York:Academic Press, 1996.
starch affinity site," Agricultural and Biological Chemistry Wyss, M. et al. "Biochemical Characterization of Fungal Phytases
53(4):923-929, 1989. (myo-lnositol Hexakisphosphate Phosphohydrolases):Catalytic
Jensen, B. et al. "Purification of extracellular arnylolytic enzymes Properties," Appl. Environ. Microbial. 65(2):367-373, Feb. 1, 1999.
Yarnada, K. et al. "Phytase fromAspergillus terreus,"Agr. Biol. Chem
from the thermophilic fungus Thermomyces lanuginosus," Can. J
32(10):1275-1282, 1968.
Microbial. 34(3):218-223, 1988.
Yoon, Seong Jun et al. "Isolation and identification of phytase-pro
Miller, G.L. "Use ofDinitrosalicylic Acid Reagent for
ducing bacterium, Enterobacter sp. 4, and enzymatic properties of
Determination of Reducing Sugar," Anal. Chem. 31(3):426-428,
phytase enzyme," Enzyme and Microbial Technology 18(6):449-
Mar. 1, 1959.
454, May 1, 1996.
Shibuya, I. et al. "Molecular cloning of the glucoarnylase gene of
Aspergillus shirousami and its expression in Aspergillus oryzae," * cited by examiner
Agricultural and Biological Chemistry 54(8):1905-1914, Aug. 1990.
 1-· - •·" .:4 water r:J)_
•
a-Amylase k: ·::··=- =-=::·x::::-::::====--·::::--::-::::--91·1 -M-il-le-
d--,
Granular Starch
,- 1 1 Grain
Hydrolyzing Enzymes =
I I Yeast
I ,
:::=====::: Nutrients
Backset. 85 C
"':I
H
c;)

......

2'
Starch ?
Fermentation N
Modification pH: 3 - 5 CIO
N
DS: 20-40% T: 25 - 35 C 0
pH: 4 - 6
Hrs:24 - 72 ETHANOL ....
T: 55 - 70 C
Hrs: 2 - 24

I -: ,·, _-- ·-c= d

c==i r,r;_
-....l
\0
Substrate Prep. Tank Fermentor Distillation 0--,

column Distillers Grains & 00

Solubles
w
"00'""'
=
N
 US 7,968,318 B2
1 2
PROCESS FOR CONVERSION OF cereal grain and combining the ground cereal grain with
GRANULAR STARCH TO liquid to form a slurry which is then mixed with one or
ETHANOL more granular starch hydrolyzing enzymes and optionally
yeast at temperatures below the granular starch
FIELD OF THE INVENTION gelatinization tem-
5 perature to produce ethanol and other co-products (U.S. Pat.
The present invention relates to processes for the produc No. 4,514,496, WO 03/066826; WO 04/081193; WO
tion of an alcohol (e.g., ethanol) from a granular starch 04/106533; WO 04/080923 and WO 05/069840).
com prising exposing a slurry comprising granular starch While the above mentioned fermentation processes using
from plant material to an alpha-amylase at a temperature a milled grain slurry in combination with granular
below the gelatinization temperature of the granular starch starch
followed by fermentation with a fermenting microorganism. 10 hydrolyzing enzymes offer certain improvements over previ
ous processes, additional fermentation process improve
BACKGROUND OF THE INVENTION ments are needed by the industry for the conversion of
granu lar starch resulting in higher energy efficiency and
The commercial viability of producing ethanol as a fuel high end product production. The object of the present
source from agricultural crops has generated renewed invention is to
world wide interest due to a variety of reasons that include 15 provide improved processes for the conversion of granular
contin ued and increased dependence on limited oil supplies starch into alcohol (e.g. ethanol) and other end products.
and the fact that ethanol production is a renewable energy
source. SUMMARY OF THE INVENTION
Alcohol fermentation cooled down and further
production processes and treated with saccharifying
particu- 20 larly ethanol enzymes (e.g. glucoamylases)
production processes are to produce fermentable
generally character ized as wet glucose. The mash containing
milling or dry milling glucose is then fermented for
processes. Reference is made approximately 24 to 45 120
to Bothast et al., 2005,Appl. hours in the presence of
Microbial. Biotechnol. ethanol producing
67:19-25 and THE ALCOHOL microorgan isms. The solids
TEXTBOOK, 3rd Ed (K. A. in the mash are separated
Jacques et al. Eds) 1999 from the liquid phase and
Nottingham University Press, ethanol and useful co-
UK for 25 products such as distillers'
a review of these processes. grains are obtained.
In general, the wet milling Improvements to the above
process involves a series of fermentation processes have
soaking (steeping) steps to 50 been accomplished by
soften the cereal grain wherein combining the
soluble starch is removed saccharification step and
followed by recovery of the fermentation step in a process
germ, fiber (bran) and gluten referred to as simultaneous
(protein). The remaining starch saccharification and
is 30 further processed by fermentation or simultaneous
drying, chemical and/or sacchari fication, yeast
enzyme treat ments. The starch propagation and fermentation.
may then be used for alcohol These improved fermentation
production, high fructose corn processes have advantages
syrup or commercial pure over the previously 55
grade starch. described dry milling
In general, dry grain milling fermentation or even wet
involves a number of basic milling fer mentation
steps, which include: grinding, processes because significant
cooking, liquefaction, saccha- sugar concentrations
35 rification, fermentation and do not develop in the
separation of liquid and solids fermenter thereby avoiding
to produce alcohol and other sugar inhi bition of yeast
co-products. Generally, whole growth. In addition, bacterial
cereal, such as corn cereal, is growth is reduced due to lack
ground to a fine particle size of easily available glucose.
and then mixed with liquid in a Increased 60 ethanol
slurry tank. The slurry is production may result by use
subjected of the simultaneous
to high temperatures in a jet saccharification and
cooker along with liquefying 40 fermentation processes.
enzymes (e.g. alpha-amylases) More recently,
to solubilize and hydrolyze fermentation processes have
the starch in the cereal to been intro duced which
dextrins. The mixture is eliminate the cooking step or
 US 7,968,318 B2
1 2
which reduce the need for The present invention granular starch; and for a
treating cereal grains at high provides processes for periodof5 minutes to 24
temperatures. These 65 producing an alcohol (e.g. hours and obtaining a
fermentation processes ethanol) from granular mash substrate
which are sometimes starch by contacting the comprising greater than
referred to as granular starch with an 20% glucose, and
no-cook, low temperature or alpha-amylase and b) fermenting the substrate
warm cook, include milling providing suitable in the presence of a
of a conditions for endogenous fermenting
plant hydrolytic enzymes, microorganism and a
which hydrolyze starch hydrolyzing
solubilized starch to enzyme at a temperature
produce glucose. The of between 10° C. and
glucose may then be used 40° C. for a period of 10
as a feedstock in hours to 250 hours to
fermentations to produce produce ethanol.
alcohol. In further embodiments of
In one aspect, the either aspect described above,
invention relates to a the process includes
process of produc ing recovering the ethanol. In yet
glucose from a granular further embodiments of the
starch substrate described aspects, the process
comprising: may include additional steps
a) contacting a slurry not specified which are
comprising granular performed prior to, during or
starch obtained from after the enumerated steps.
plant material with an
alpha-amylase at a BRIEF DESCRIPTION
tempera ture below OF THE DRAWINGS
the starch
gelatinization FIG. 1 is a general
temperature of the schematic diagram that
granular starch to illustrates an embodiment of
produce the invention wherein the
oligosaccharides and slurry comprising a milled
allow ing endogenous grain containing granular
plant carbohydrate starch and having a DS of 20
hydrolyzing enzymes to 40% is contacted with an
to hydrolyze the alpha-amylase at a
oligosaccharides, and temperature between 55° C. to
b) producing a mash 70° C. and a pH of 4.0 to 6.0
comprising at least 10% for 2 to 24 hours. The
glucose. resulting mash comprising
In a further embodiment glucose is transferred
of this aspect, the mash is
fer mented in the presence
of a fermenting
microorganism and starch
hydrolyzing enzymes at a
temperature of between
10°
C. and 40° C. for a period
of time of 10 hours to 250
hours to produce alcohol,
particularly ethanol.
In another aspect, the
invention relates to a
process for producing
ethanol comprising:
a) contacting a slurry
comprising granular
starch with an alpha-
amylase capable of
solubilizing granular
starch, wherein said
contacting is at a pH
of 3.5 to 7.0; at a
temperature below the
starch gelatinization
temperature of the
 US 7,968,318 B2
3 4
to a fermentor and fermented at pH 3.0 to 5.0 at a those effecting the exo or endohydrolysis of 1,4-a-D-gluco
temperature of25° C. to 35° C. for 24 to 72 hours in the sidic linkages in polysaccharides containing 1,4-a-linked D-
presence of yeast, nutrients, acid and starch hydrolyzing glucose units.
enzymes to produce ethanol. The term "gelatinization" means solubilization of a starch
5 molecule by cooking to form a viscous suspension.
DETAILED DESCRIPTION OF THE INVENTION The term "gelatinization temperature" refers to the lowest
temperature at which gelatinization of a starch containing
Unless defined otherwise herein, all technical and scien substrate begins. The exact temperature of gelatinization
tific terms used herein have the same meaning as commonly depends on the specific starch and may vary depending on
10
understood by one of ordinary skill in the art to which this factors such as plant species and environmental and growth
invention belongs. conditions.
Although any methods and materials similar or The term "below the gelatinization temperature" refers to a
equivalent to those described herein can be used in the temperature that is less than the gelatinization temperature.
practice or testing of the present invention, the The term "glucoamylase"
preferred methods and refers to the
materials are described. amyloglucosidase
The invention will now class of enzymes (e.g.,
be described in detail by E.C.3.2.1.3,glucoamylase,
way of reference only using 1,4-alpha D-glucan
the following definitions glucohydrolase). These are
and examples. All patents exo-acting enzymes, which
and publications, including release glucosyl residues
all sequences dis from the non-reducing ends
of amylase and amylopectin
molecules. The enzymes
also
closed within such patents 20 hydrolyzes alpha-1,6 and
and publications, referred to alpha-1,3 linkages although
herein
at much slower rate than
are expressly
alpha-1,4 linkages.
incorporated by
The phrase
reference.
"simultaneous
Definitions saccharification and
The term "fermentation" fermenta tion (SSF)" refers
refers to the enzymatic and to a process in the
anaerobic breakdown of production of end prod ucts
organic substances by in which a fermenting
microorgan isms to produce organism, such as an
simpler organic compounds. ethanol
While fermen tation occurs 25 producing microorganism,
under anaerobic conditions it and at least one enzyme,
is not intended that the term such as a saccharifying
be solely limited to strict enzyme are combined in
anaerobic conditions, as the same process step in the
fermentation also occurs in same vessel.
the presence of oxygen. The term
As used herein the term "saccharification"refers to
"starch" refers to any enzymatic conversion of a
material directly unusable
polysaccharide to a mono-
or oligosac
comprised of the complex aqueous mixture
polysaccharide carbohydrates comprising
of 30 plants, comprised of insoluble solids, (e.g. granular
amylase and amylopectin with starch).
the for mula (C6 H100 5 )x, The term "dextrins" refers
wherein x can be any number. to short chain polymers of
The term "granular starch" glu
refers to raw (uncooked) cose (e.g. 2 to 10 units).
starch, The term
that is starch in its natural form "oligosaccharides" refers to
found in plant material (e.g. any compound hav ing 2 to
grains and tubers). 10 monosaccharide units
As used herein the term "dry joined in glycosidic link ages.
solids content (DS)" refers to These short chain polymers of
the total solids of a slurry in% simple sugars include 45
on a dry weight basis.
dextrins.
The term "slurry" refers to an The term "soluble starch"
 US 7,968,318 B2
3 4
refers to starch which charide for fermentative As used herein the term product. As used herein the
results conversion to an end "fermenting organism" refers to term "ethanol producer" or
from the hydrolysis of product. any microorganism or cell ethanol producing
insoluble starch (e.g. The term "milling" which is suitable for use in fer microorganism" refers to a
granular starch). The refers to the breakdown of mentation for directly or fermenting organism
term "mash" refers to a cereal grains to smaller indirectly producing an end
mixture of a particles. In some and "enzymes having 60 that is capable of producing
fermentable embodiments the term is granular starch hydrolyzing ethanol from a mono- or oli
used interchangeably with (GSH) activity" refer to gosaccharide.
grinding. enzymes, which have the The term "derived"
The term "dry milling" ability to hydro lyze starch in encompasses the terms
refers to the milling of dry granular form. "originated from",
whole grain, wherein The term "hydrolysis of "obtained" or "obtainable
fractions of the grain such starch" refers to the cleavage from", and "isolated from"
as the germ and bran have of glucosidic bonds with the and in some embodiments
not been purposely addition of water molecules. as used herein means that a
removed. The term "alpha-amylase 65 polypeptide encoded by the
The term "liquefaction" (e.g., E.C. class 3.2.1.1)" nucleotide sequence is
refers to the stage in starch refers to enzymes that produced from a cell in
con version in which catalyze the hydrolysis of which the nucleotide is
gelatinized starch is alpha-1,4-gluco sidic naturally present or in
hydrolyzed to give low linkages. These enzymes have which the nucleotide has
molecular weight soluble also been described as been inserted.
dextrins.
The term "thin-stillage"
refers to the resulting
liquid por tion of a
fermentation which
contains dissolved material
and suspended fine
particles and which is
separated from the solid
portion resulting from the
fermentation. Recycled
thin-still- age in industrial
fermentation processes is
frequently referred to as
"back-set".
The term "vessel"
includes but is not limited
to tanks, vats, bottles,
flasks, bags, bioreactors
and the like. In one
embodi ment, the term
refers to any receptacle
suitable for conducting the
saccharification and/or
fermentation processes
encom-
substrate in liquid used in 50 passed by the invention.
the production of a The term "end
fermented product and is product" refers to any
used to refer to any stage of carbon-source derived
the fermentation from the product which is
initial mixing of the enzymatically converted
fermentable substrate with from a fermentable
one or more starch substrate. In some
hydrolyzing enzymes and preferred embodiments,
fermenting organ isms the end product is an
through the completion of alcohol, such as ethanol.
the fermentation run.
The terms "saccharifying enzyme"
enzyme" and "starch
hydrolyz- 55 ing enzymes"
refer to any enzyme that is
capable of convert ing starch
to mono- or oligosaccharides
(e.g. a hexose or pentose).
The terms "granular starch
hydrolyzing (GSH)
 US 7,968,318 B2
5 6
The term "heterologous" with reference to a protein or entire plant may be used, for example, the entire corn stover
polynucleotide refers to a protein or polynucleotide that may be used. In one embodiment, whole grain may be used
does not naturally occur in a host cell. as a source of granular starch. Preferred whole grains
The term "endogenous" with reference to a protein or include corn, wheat, rye, barley, sorghum and combinations
polynucleotide refers to a protein or polynucleotide that does 5 thereof. In other embodiments, granular starch may be
naturally occur in a host cell. obtained from fractionated cereal grains including fiber,
The phrase "endogenous plant hydrolytic enzymes endosperm and/or germ components. Methods for
capable of hydrolyzing soluble starch" refers to hydrolytic fractionating plant material such as com and wheat are
enzymes that are expressed and produced in a plant and known in the art. In some embodi ments, plant material
may be pro obtained from different sources may be mixed together to
duced by the expression of endogenous or heterologous 10 obtain granular starch used in the processes of the invention
genes. (e.g. corn and milo or corn and barley).
The term "enzymatic conversion" in general refers to the In some embodiments, plant material comprising granular
modification of a substrate by enzyme action. The term as starch may be prepared by means such as milling. In
used herein also refers to the modification of a fermentable particu lar, means of milling whole cereal grains are well
known and
substrate, such as a granular 15 include the use of hammer
starch containing substrate mills and roller mills.
by the action of an enzyme. Alpha-Amylases-
The terms "recovered", In some of the
"isolated", and "separated" embodiments
as used herein refer to a encompassed by the
compound, protein, cell, inven tion, the alpha-
nucleic acid or amino acid amylase is a microbial
that is removed from at least enzyme having an E.C.
one component with which number, E.C. 3.2.1.1-3
it is naturally associated. and in particular E.C.
As used herein the term 3.2.1.1. Any
"enzyme unit" refers to the 20 suitable alpha-amylase
amount of enzyme that may be used in the
present process. In some
produces 1 micromole of
embodiments, the alpha-
product per minute under
amylase is derived from a
the specified conditions of
bac terial strain and in
the assay. For example, in
other embodiments the
one embodiment, the term
alpha-amylase is derived
"glucoamylase activity unit"
from a fungal strain. In
(GAU) is defined as the
further embodiments, the
amount of enzyme required preferred alpha-amylase
to pro duce 1 g of glucose is a bacterial alpha-
per hour from soluble starch amylase. In other
substrate (4% DS) under 25 embodiments, the alpha-
assay conditions of 60° C. amylase is an acid stable
and pH 4.2. alpha-amy lase. Suitable
The term "yield" refers to alpha-amylases may be
the amount of end naturally occurring as
product pro
well as recombinant
duced using the methods of
(hybrid and variants) and
the present invention. In
mutant alpha amylases
some embodiments, the
(WO 99/19467 and WO
term refers to the volume of
97/41213). In particularly
the end product and in other
preferred embodiments,
embodiments, the term
the alpha-amylase is
refers to the concentra tion
derived from a
of the end product.
30 Bacillus species. Preferred
The term "DE" or
Bacillus species include
"dextrose equivalent" is an
B. subti lis, B.
industry standard for
stearothermophilus, B.
measuring the concentration
lentus, B. licheniformis,
of total reducing sugars,
B. coagulans,and B.
calculated as D-glucose on
amyloliquefaciens (U.S.
a dry weight basis. Unhy
Pat. Nos. 5,093, 257;
drolyzed granular starch has
5,763,385; 5,824,532;
a DE that is essentially O
5,958,739; 6,008,026,
and D-glucose has a DE of
6,361,809; 6,867,031;
100.An instructive method
WO 96/23874; WO
for deter mining the DE of a
96/39528 and WO
slurry or solution is
35 05/001064). Particularly
described in Schro orl's
preferred alpha-amylases
method (Fehling's assay
are derived from Bacillus
titration).
 US 7,968,318 B2
5 6
strains B. Pat. Nos. 6,187, 576; e
stearothermophilus, B. 6,093,562; 5,958,739; US y
amy loliquefaciens and 2006/0014265 and WO
B. licheniformis ((U.S. 99/19467). R
As used herein the term Most preferred alpha- e
"comprising" and its amylases are amylases s
cognates are 40 derived from e
used in their inclusive sense; B. stearothermophilus and a
that is, equivalent to the term B. licheniformis including r
"including" and its wild type, hybrid and variant c
corresponding cognates. alpha-amylase enzymes. See h
"A", "an" and "the" include Suzuki et al., (1989) J. Biol. )
plural references unless the Chem. 264:18933-18938 .
context clearly dictates and US 2006/ P
0014265,particularly SEQ l
otherwise.
ID NOs: 3, 4 and a
16.Reference is n
Numeric ranges are 45 also made to strains having t
inclusive of the numbers American Type Culture Collec E
defining the tion (ATCC) numbers- n
range. ATCC 39709; ATCC z
The headings provided 11945; ATCC 6598;ATCC y
herein are not limitations of 6634;ATCC 8480;ATCC m
the various aspects or 9945 andNCIB 8059. e
embodiments of the In addition to the s
invention, which can be had bacterial alpha-amylases, -
by reference to the fugal alpha amylases are Plants have naturally
specification as a whole. contemplated for use in occurring starch
the processes of the degrading enzymes
Embodiments 50 invention. Suitable fungal such as alpha-amylases
of the alpha-amylases are derived (EC 3.1.1.1); beta-
Invention from filamentous fungal amylases (EC 3.1.1.2),
strains such as Aspergillus, amyloglucosidases
(A) Raw Materials: such as A. oryzae and A. (glucoamylase)(EC
Granular Starch- niger (e.g. FUNGAMYL 3.1.1.3) and
Granular starch may be and CLARASE L), and 65 starch phosphorylases
obtained from plant material Trichoderma, Rhizopus, (EC 2.4.1.1). In
including but not limited to Mucor, and Penicillium. addition, plants may
wheat, com, rye, sorghum Commercially available have been genetically
(milo), rice, millet, barley, alpha-amylases engineered to include
triticale, cassava (tapioca), contemplated for heterologous genes
potato, sweet potato, sugar 55 use in the methods of the encoding starch
beets, sugarcane, and invention include; degrading enzymes,
legumes such as soybean and SPEZYME AA; such as amylases,
peas. Preferred plant material SPEZYME FRED;
includes corn, barley, wheat, SPEZYME ETHYL;
rice, milo and combinations GZYME G997;
thereof. Particularly pre CLARASE L (Genencor
ferred plant material is International Inc.);
obtained from corn (Zea TERMAMYL 120-L, LC,
mays). Plant material may SC and SUPRA
include hybrid varieties and (Novozymes Biotech);
genetically modi fied LIQUOZYME X and
varieties (e.g. transgenic SAN SUPER (Novozymes
com, barley or soybeans A/S) and
60
com prising heterologous
genes). Any part of the plant U
may be used to provide L
T
granular starch including but
R
not limited to plant parts
A
such as leaves, stems, hulls,
husks, tubers, cobs, grains
T
and the like. In some H
embodiments, essentially the I
N

(
N
a
l
l
 US 7,968,318 B2
7 8
glucoamylase and others (WO 03/018766 and WO ary enzymes. In other embodiments, the additional enzymes
05/096804). Endogenous starch degrading plant enzymes, will be included in the fermentation step along with yeast
whether naturally occurring or expressed from an introduced and other components.
polynucleotide, with exposure to elevated temperatures, such In some embodiments during the contacting step with the
as the temperatures of jet cooking or even temperatures above alpha-amylase, the secondary enzyme may include a glu
5 the gelatinization temperature of granular starch will become coamylase, granular starch hydrolyzing enzymes, a protease,
inactivated. However, at temperatures conducted in the a phytase, a cellulase, a hemicellulases, a pullulanase, a
present process, it is believed that the endogenous starch xyla nase, a lipase, a cutinase, a pectinase, a beta-glucanase,
degrading enzymes are not inactivated and actually contrib a beta amylase, a cyclodextrin transglycosyltransferase and
combi- nations thereofln some preferred embodiments, the
ute to the hydrolysis of granular starch. Although not bound to
10 contact ing step will include a combination of an alpha-
amylase, a phytase and optionally a protease. In other
theory, the inventors believe that the alpha-amylase
embodiments, the contacting step will include a
provided in the contacting step modifies the granular starch combination of an alpha-amy lase, a glucoamylase and
structure of the plant material allowing for the production of optionally a protease. In yet other embodiments, the
oligosaccha contacting step will include a combination of an alpha-
rides including dextrins. The oligosaccharides are further 15 amylase, a glucoamylase, a phytase and option- ally a
degraded at the temperatures encompassed by the contacting protease.
step (e.g. 45° C. to 70° C.) by plant starch degrading enzymes. Glucoamylases (GA) (E.C. 3.2.1.3.) may be derived from
The plant starch degrading enzymes act on the partially the heterologous or endogenous protein expression of bacte
hydrolyzed starch to produce glucose. While exogenous ria, plants and fungi sources. Preferred glucoamylases
sources of glucoamylases may be included in the contacting 20 useful in the compositions and methods of the invention are
step, the addition of exogenous glucoamylase is not required pro- duced by several strains of filamentous fungi and yeast.
to provide glucose, which is then optionally used as a feed In particular, glucoamylases secreted from strains of
stock for alcohol fermentation. Therefore in one embodi Aspergil lus and Trichoderma are commercially important.
ment, the contacting step of the invention does not include Suitable
the addition of glucoamylases or in other embodiments the
derived from microbial microorganism may be a
sources. However, the recombinant microorganism.
addition of glucoamylases For example, in some
and/or other enzymes such embodiments the preferred
as phytases and proteases fermenting microorganisms
may increase the production include bacterial strains from
of solubilized granular Bacillus, Lactobacillus,
starch. E.coli, Erwinia, Pantoea (e.g.,
Fermenting Organisms- P. citrea) and Klebsiella (e.g.
Examples of fermenting K. oxy toca). (See e.g. U.S.
organisms are Pat. Nos. 5,028,539,
ethanologenic 5,424,202 and WO 95/13362).
microorganisms or ethanol In further preferred
producing microorganisms embodiments, the ethanol-
such as ethanologenic producing microorganism is a
bacteria which express fungal microorganism, such as
alcohol dehydroge nase and a yeast and specifically
pyruvate dehydrogenase Saccharomyces such as strains
and which can be obtained of S. cerevisiae (U.S. Pat. No.
from Zymomonas moblis 4,316,956). A variety of S.
(See e.g. U.S. Pat. Nos. cerevisiae are com mercially
5,000,000; 5,028,539, available and these include
5,424,202; 5,514,583 and but are not limited to PALI
5,554,520). In addi (Fleischmann'sYeast),
tional embodiments, the SUPERSTART (Alltech),
ethanologenic FER MIOL (DSM
microorganisms express Specialties), RED STAR
xylose reductase and xylitol (Lesaffre) and Angel alcohol
dehydrogenase, enzymes yeast (Angel Yeast Company,
that convert xylose to China).
xylulose. In further embodi Secondary Enzymes-
ments, xylose isomerase is While in one embodiment,
used to convert xylose to it is contemplated that addi
xylulose. In particularly tional starch hydrolyzing
preferred embodiments, a enzymes are not needed, and
microorganism capable of there fore not included in the
fermenting both pentoses contacting step, additional
and hexoses to ethanol are enzymes may be included in
utilized. For example, in both the contacting step and
some embodiments the fermenting step encompassed
micro organism may be a by the invention. In some
natural or non-genetically embodiments, these enzymes
engineered microorganism will be included as a
 US 7,968,318 B2
7 8
secondary enzyme in the 25 glucoamylases include rolfsii and vari ants
contacting step, which naturally occurring wild- thereof (WO
comprises contacting the type glu coamylases as 04/111218).
granular starch slurry with an well as variant and Enzymes having
alpha-amylase and one or genetically engineered glucoamylase activity
more second- mutant glucoamylases. The used commercially
following glucoamylases 55 are produced for
are nonlimiting examples example,
of glucoamylases that may fromAspergillus niger
be used in the process (trade name
encompassed by the DISTILLASE,
invention. Aspergillus OPTIDEX L-400 and G
niger ZYME G990 4X from
30 Gl and G2 glucoamylase Genencor International
(Boe! et al., (1984) EMBO Inc.) or Rhizopus
J. 3:1097-1102; WO species (trade name
92/00381, WO00/04136 CU.CONC from Shin
and U.S. Pat. No. Nihon Chemicals,
6,352,851); Aspergillus Japan). Also the
awamori glucoamylases commercial digestive
(WO 84/02921); enzyme, trade name
Aspergillus oryzae GLUCZYME
glucoamylases (Hata et al., 60 from Amano
(1991) Agric. Biol. Chem. Pharmaceuticals, Japan
55:941-949) and (Takahashi et al., (1985)
Aspergillus shi- J. Biochem. 98:663-
35 rousami. (See Chen et al., 671). Additional
(1996) Prat. Eng. 9:499- enzymes include three
505; Chen et al. (1995) forms of glucoamylase
Prat. Eng. 8:575-582; and (E.C.3.2.1.3) of a
Chen et al., (1994) Rhizopus sp., namely
Biochem J. 302:275-281). "Glucl" (MW 74,000),
Glucoamylases are also "Gluc2" (MW 58,600)
obtained from strains of and "Gluc3" (MW
Talaro myces such as those 61,400). Also the
derived from T. emersonii, enzyme preparation
T. leycettanus, GC480
40 T. duponti and T. 65 (Genencor International
thermophilus (WO Inc.) finds use in the
99/28488; U.S. Pat. No. invention.
RE: 32,153; U.S. Pat. No. Granular starch
4,587,215); strains hydrolyzing enzymes
ofTrichoderma, such as T. (GSHEs) are able to
reesei T reesei and hydrolyze granular
particularly glucoamylases starch, and these
having at least 80%, 85%, enzymes have been
90% and 95% sequence
identity to SEQ ID NO: 4
disclosed in US Pat. Pub.
No. 2006-0094080;
45 strains of Rhizopus, such as
R. niveus andR. oryzae;
strains of Mucor and
strains of Humicola, such
as H. grisea (See, Boe! et
al., (1984) EMBO J.
3:1097-1102; WO
92/00381; WO 00/04136;
Chen et al., (1996) Prat.
Eng. 9:499-505; Taylor et
al., (1978) Carbohydrate
Res. 61:301-308; U.S.
Pat. Nos.
50 4,514,496; 4,092,434; and
Jensen et al., (1988) Can.
J. Micro bial. 34:218-223).
Other glucoamylases
useful in the present
invention include those
obtained fromAthelia
 US 7,968,318 B2
9 10
recovered from fungal, bacterial and plant cells such as Bacil- teases such as NSP24 and also GC106 (Genencor Interna
lus sp., Penicillium sp., Humicola sp., Trichoderma sp. tional Inc.). Preferred fungal proteases are derived from
Aspergillus sp. Mucor sp. and Rhizopus sp. In one embodi strains of Aspergillus (e.g. proteases from A. niger and A.
ment, a particular group of enzymes having GSH activity oryzae), Mucor (e.g. M. miehei), Trichoderma,
include enzymes having glucoamylase activity and/or alpha- 5 Rhizopus,and Candida. Preferred bacterial proteases are
amylase activity (See, Tosi et al., (1993) Can. J. Microbial. derived from strains of Bacillus such as B.
39:846-855). ARhizopus oryzae GSHE has been described in amyloliquefaciens.Proteases added to the fermentation may
Ashikari et al., (1986) Agric. Biol. Chem. 50:957-964 and increase the free amino nitro gen level and increase the rate
U.S. Pat. No. 4,863,864. A Humicola grisea GSHE has been of metabolism of the yeast and further give higher
described in Allison et al., (1992) Curr. Genet. 21:225-229; 10 fermentation efficiency.
WO 05/052148 and European Patent No. 171218. An Enzymes that may be used in the methods of the
Aspergillus awamori var. Kawachi GSHE has been described invention include beta-amylases (E.C. 3.2.1.2). These are
by Hayashida et al., (1989) Agric. Biol. Chem 53:923-929. exo-acting maltogenic amylases, which catalyze the
An Aspergillus shirousami GSHE has been described by hydrolysis of 1,4- alpha-glucosidic linkages in amylase,
amylopectin and related glucose polymers. Commercial
beta-amylases are
Shibuya et al., (1990) Agric. 15 available from Genencor
Biol. Chem. 54:1905-1914. International Inc., and
In one embodiment, a examples include
GSHE may have SPEZYME BBA and
glucoamylase activity and is OPTIMALT BBA.
derived from a strain of Cellulases (E.C.
Humicola grisea, 3.2.1.4) such as endo-
particularly a strain of glucanases may be used
Humicola grisea var. in the methods of the
thermoidea (see, invention. Examples of
U.S. Pat. No. 4,618,579). cellulases include
In some preferred cellulases from
embodiments, filamentous fungus such
as Tricho
the Humicola enzyme having derma, Humicola,
GSH activity will have at Fusarium,and Aspergillus.
least 20 Commercially cellulases
85%, 90%, 92%, 94%, 95%, are available as SPEZYME
96%, 97%, 98% and 99% CP and LAMINEX
sequence identity to the (Genencor International,
amino acid sequence of Inc) and CELLUZYME
SEQ ID NO: 3 of WO and ULTRAFLO
05/052148. (Novozymes A/S).
In another embodiment, a Xylanases useful in the
GSHE may have methods of the invention
glucoamylase may be
activity and is derived from 25 from bacterial or fungal
a strain of Aspergillus sources, such as
awamori, particularly a Aspergillus, Tricho
strain of A. awamori var. derma, Neurospora,
kawachi. In some preferred andFusarium.
embodiments, the A. Commercial preparations
awamori var. kawachi include SPEZYME CP
enzyme having GSH and LAMINEX
activity will have at least (Genencor Interna tional,
85%, 90%, 92%, 94%, Inc.) and ULTRAFLO
95%, 96%, 97%, 98% and (Novozymes A/S).
99% sequence identity to A number of bacterial
the amino acid sequence of and fungal phytases
SEQ ID NO: 6 of WO (E.C. 3.1.3.8
05/052148. 30 and 3.1.3.26) are known
In another embodiment, a and in some embodiments
GSHE may have the addi tion of phytases
glucoamylase activity and is are particularly useful in
derived from a strain of the methods. Yeast
Rhizopus,such as R. niveus phytases may be derived
or R. oryzae. The enzyme from strains of
derived from the Koji strain Saccharomyces (e.g.
R. niveus is sold under S. cerevisiae) and
the trade name "CU CONC Schwanniomyces (e.g. S.
or the occidentalis)
(Wadzinski et al., Adv.
Apple. Microbial.,
42:263-303).
 US 7,968,318 B2
9 10
enzyme from Rhizopus sold Other fungal phytases have 40 WO 98/28409; WO alpha-amylase is conducted
under the trade name 35 been described in the 97/38096 and WO at a pH
GLUZYME. literature and reference is 9844125; and U.S. Pat. 65 range of3.5 to 7.0;also at a
Anotheruseful GSHE made to Wyss et al., (1999) Nos. 6,734,004; pH range of3.5 to
having glucoamylase Appl. Environ Microbial. 6,350,602; and 6.5;preferably at a pH
activity is SPI RIZYME 65:367-373; Berka et al., 5,863,533). Fungal range of 4.0 to 6.0 and
Plus (Novozymes A/S), (1998) Appl. Environ. phytases have been more preferably at a pH
which also includes acid Microbiol. 64: 4423-4427; derived range of 4.5 to 5.5.The
fungal amylase activity. Yamada et al., (1986) Agric. fromAspergillus (e.g. slurry is held in contact
Biol. Chem. 322:1275-1282; A. niger, A. awamori, with the alpha-
PCT Publication Nos. WO A. terreus, A. oryzea
98/28408; and A. fumigatus);
In another embodiment, a microbial proteases, such as Thermo myces
GSHE may have alpha- fungal and bacterial proteases, (Humicola)
amylase activity and is for example, acid fungal pro- lanuginousus;
derived from a strain of Fusarium (F.
Aspergillus such as a strain javanicum
of A. awamori, A. niger, A. 45 and F. versillibodes).
oryzae, or A. kawachi and Bacterial phytases may
particularly a strain of A. also find use in the
kawachi. invention (Greiner R. et
In some preferred al. (1993) Arch.
embodiments, the A. Biochem. Bio phys.
kawachi enzyme having 303: 107-113; Yoon S.
GSH activity will have at J. et al. (1996) Enzyme
least 85%, 90%, 92%, 94%,
and Microbial Technol.
95%, 96%, 97%, 98% and 18: 449-454; and WO
99% sequence identity to the 06/043178).
amino acid sequence of SEQ Commercially
ID NO: 3 ofWO 05/118800 available phytases
and WO 05/003311. which may be used
In some embodiments, the 50 according to the
enzyme having GSH activity invention include
is a hybrid enzyme, for PHYZYME XP 5000
example one containing a (Da nisco A/S);
catalytic domain of an alpha- FINASE (Altech); GC
amylase such as a catalytic 491; FINASE,
domain of an Aspergillus SPEZYME HPA
niger alpha-amylase, an (Genencor), BIO-FEED
Aspergillus oryzae alpha- PHYTASE and
amylase or an Aspergillus PHYTASE NOVO
kawachi alpha-amylase and a (Novozymes) and
starch binding domain of a NATUPHOS (DSM).
different fungal alpha- One skilled in the art
amylase or glucoamylase, can readily determine
such as an Aspergillus the effective
kawachi or a Hurni cola 55 amount of the enzymes
grisea starch binding which may be used in
domain. In other the process steps
embodiments, the hybrid encompassed by the
enzyme having GSH activity invention.
may include a catalytic (B) Process Steps-
domain of a glucoamylase, The granular starch
such as a catalytic domain of (e.g. milled cereal
an Aspergillus sp., a grain) to be pro cessed
Talaromyces sp., an Althea is mixed with an
sp., a Tricho derma sp. or a aqueous solution to
Rhizopus sp. and a starch obtain a slurry.
binding domain of a different 60 The aqueous solution
glucoamylase or an alpha- may be obtained, for
amylase. Some hybrid example from water,
enzymes having GSH thin stillage and/or
activity are disclosed in WO backset.
05/003311, WO 05/045018; A slurry may have a
Shibuya et al., (1992) Biosci. DS of between 5-60%;
Biotech. Biochem 56: 1674- 10-50%; 15-45%; 15-
1675 and Cornett et al., 30%; 20-45%; 20-
(2003) 30% and also 25-40%.
Protein Engineering 16:521- The
520. contacting step with an
Suitable proteases include
 US 7,968,318 B2
11 12
amylase at a temperature below the starch gelatinization tem substrate (mash) is obtained which comprises greater than
perature of the granular starch. In some embodiments, this 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,
temperature is held between 45° C. and 70° C.; in other 60%, 65%, 70%, 75% and 80% glucose.
embodiments, the temperature is held between 50° C. and 70° In some preferred embodiments of the contacting step, a
C.; between 55° C. and 70° C.; between 60° C. and 70° C., 5 slurry comprising granular corn starch having a DS of 20-
between 60° C. and 65° C.; between 55° C. and 65° C. and 40% is contacted with an alpha-amylase derived from
between 55° C. and 68° C. In further embodiments, the tem Bacillus stearothermophilus or Bacillus licheniformis for 1
perature is at least 45° C., 48° C., 50° C., 53° C., 55° C., 58° to 6 hours at a temperature between 55° C. to 70° C. to
C., 60° C., 63° C., 65° C. and 68° C. In other embodiments, obtain a soluble starch substrate comprising at least 30%
the temperature is not greater than 65° C., 68° C., 70° C., 73° 10 glucose. In other preferred embodiments of the contacting
C., 75° C. and 80° C. step, a slurry comprising granular milo starch having a DS
The initial starch gelatinization temperature ranges for a of 20-40% is contacted with an alpha-amylase derived from
number of granular starches which may be used in accor dance Bacillus stearothermophilus or Bacillus licheniformis for 1
with the processes herein include barley (52° C. to 59° C.), to 6 hours at a temperature between 55° C. to 70° C. to
wheat (58° C. to 64° C.), rye (57° C. to 70° C.), corn (62° 15 obtain a soluble starch substrate comprising at least 50%
C. to 72° C.), high amylase corn (67° C. to 80° C.), rice (68° glucose.
C. to 77° C.), sorghum (68° C. to 77° C.), potato (58° C. to 68° After the contacting step which results in the production
C.), tapioca (59° C. to 69° C.) and sweet potato (58° C. to 72° of a mash comprising glucose, the mash is subjected to
C.). (J. J.M. Swinkels pg 32-38 in STARCH CONVERSION fermen tation with a fermenting microorganism (e.g. an
TECHNOLOGY, EdsVanBeynumetal., (1985)Marce1Dek- 20 ethanol producing microorganism).
ker Inc. New York and The Alcohol Textbook 3rd ED. A However, prior to subjecting the mash including at least
Reference for the Beverage, Fuel and Industrial Alcohol 10% glucose to fermentation, the mash may be further
Industries, Eds Jacques et al., (1999) Nottingham University exposed to an aqueous solution comprising, for example
Press, UK). backset and/or corn steep 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
embodi-
In the contacting step, the GAU/g DS; also 0.05 to 10
slurry may be held in GAU/gDS; also0.1 to
contact with the alpha- l0GAU/gDS andeven0.5to 5
amylase for a period of 5 GAU/g DS.
minutes to 48 hours; and In some embodiments, the
also for a period of 5 effective dose of a phytase to
minutes to 24 hours. In be used in the contacting step
some embodiments the and/or fermentation step will
period of time is between 15 be in the range of0.001 to 15
minutes and 12 hours, 15 FTU/g DS; also 0.005 to 10
minutes and 6 hours, 15 FTU/g DS; and also 0.05 to 5
minutes and 4 hours and FTU/g DS. One phytase unit
also 30 minutes and 2 (FTU) is
hours.
The effective
concentration of the alpha-
amylase used in the
contacting step will vary
according to the specific
process conditions and
granular starch used.
However, in general the
amount of alpha-amylase
used will be in the range
of0.001 to 50 AAU/g DS,
0.01 to 30 AAU/g DS, 0.01
to 10 AAU/g DS
and also 0.05 to 5.0 AAU/g
DS.
In some embodiments,
the effective dose of an
alpha-amy lase in the
contacting step and/or
fermentation step will be
0.01 to 15 SSU/g DS; also
0.05 to 10 SSU/g DS; also
0.1 to 10 SSU/g DS; and 0.5
to 5 SSU/g DS.
In some embodiments,
the effective dose of a
glucoamy lase for the
contacting step and/or the
fermentation step will be in
the range of 0.01 to 15
 US 7,968,318 B2
11 12
25 ment of the invention, the comprising at least 10% 60 described above,
% DS of the mash may glucose is then subjected to fermentation media will
be diluted. For example, fermentation processes contain supple ments
the DS of the diluted using fermenting including but not
mash maybe between 5 microorganisms as limited to vitamins (e.g.
to 35%; 5 to 30%; 5 to described above. These biotin, folic acid,
25%; 5 to 20%; 5 to fermentation processes are nicotinic acid,
20%; 5 to 15%; and 5 to described in The Alcohol riboflavin), cofactors,
10% less than the % DS Text- and macro and micro-
of the slurry in the 40 book 3rd ED, A Reference nutrients and salts (e.g.
contacting step. In one for the Beverage, Fuel and (NH4)2 SO4 ; K2 HPO4 ;
non-limiting example, if Indus trial Alcohol NaCl; MgSO4 ; H3 BO3 ;
the% DS of the slurry in Industries, Eds Jacques et ZnCl2 ; and CaCl2 ) .
30 the contacting step is al., (1999) Notting ham 65 Additional enzymes to
approximately 32% and University Press, UK. be included in the
the mash is further In some preferred fermentation step
exposed to a diluting embodiments, the mash is may be the same or
aqueous solution which fermented with a yeast at different from the
dilutes the DS between 5 temperatures in the range enzymes used in the
to 10%, the DS of the of 15 to 40° C. and contacting step. In
mash to be fermented 45 also 25 to 35° C.; at a pH
some embodiments, the
will be between 22% and range of pH 3.0 to 6.5; also enzyme will
27%. In some preferred pH 3.0 to 6.0; pH 3.0 to
embodi ments, if the DS 5.5, pH 3.5 to 5.0 and also
of the contacting slurry pH 3.5 to 4.5 for a period
is between30to 35%, of time of 12 to 240 hours,
35 the DS of the diluted slurry preferably 12 to 120 and
will be about 20 to 30%. more preferably from 24 to
In a preferred 90 hours to produce an
embodiment, the mash alcohol product, preferably
ethanol.
the amount of enzyme, which Yeast cells are generally
liberates 1 micromole inor- 50 supplied in amounts ofl 04 to
ganic phosphorus per minute 1012 , and preferably from 107
from sodium phytate, 0.0051 to 1010 viable yeast count per
moles/liter, at 37° C. and at pH ml of fermentation broth.
5.0. The fermentation will
In some embodiments, the include in addition to a
effective dose of a protease to fermenting microorganisms
be used in the contacting step (e.g. yeast) nutrients, option
and/or fermentation step will ally acid and additional
be in the range of0.01 to 15 enzymes.
SAPU/g DS; also 0.01 to 10 In one preferred
SAPU/g 55 DS; and also 0.05 embodiment, the contacting
to 5 SAPU/g DS. SAPU refers step is con ducted in a
a spectro photometric acid separate vessel from the
protease unit, wherein 1 fermenting step. It is also
SAPU is the amount of contemplated that the
protease enzyme activity that contacting step and
liberates one micro mole of fermenting step may be
tyrosine per minute from a conducted in a SSF process
casein substrate under in the same vessel.
In some embodiments, in
addition to the raw materials
conditions of the assay. of time as indicated above, a
During the contacting step soluble starch
between 25-90% of the
granular starch is solubilized
to produce oligosaccharides
comprising dextrin. In some
embodiments, greater than
20%, 25%, 30%,
35%, 40%, 45%, 50%,
55%, 60%, 65%, 70%,
75%, 80%,
85% and 90% of the granular
starch is solubilized.
After contacting the
granular starch with the
alpha-amy lase for a period
 US 7,968,318 B2
13 14
include alpha-amylases and glucoamylases, including granu- STARGEN 001 (available from Genencor)-a blend of
lar starch hydrolyzing enzymes. In some preferred embodi Aspergillus niger glucoamylase and Aspergillus kawachi
ments, the glucoamylase and alpha-amylase may occur in a alpha-amylase.
blend. Particularly preferred enzyme blends include STAR The following assays were used in the examples below:
GEN 001 (Genencor International Inc.), which is a blend of 5 The activity of alpha-amylase is expressed as alpha amy
an alpha-amylase from A. kawachi and a glucoamylase from lase units (AAU) and enzyme activity was determined by
A. niger. In some preferred embodiments, the glucoamylase the rate of starch hydrolysis, as reflected in the rate of
will be derived from a Trichoderma reesei glucoamylase, a decrease of iodine-staining capacity, which was measured
Athelia rolfi glucoamylase, a Talaromyces glucoamylase, a spectrophoto metrically. One AAU of bacterial alpha-
Aspergillus glucoamylase and hybrid and variants glucoamy- amylase activity is the amount of enzyme required to
1o lase derived there from. In some preferred embodiments, the hydrolyze 10 mg of starch per min under standardized
enzyme is selected from a cellulase, a phytase and a protease. conditions.
Recovery of Alcohol and Other End Products- Alpha-amylase activity made also determined as soluble
The preferred end product of the instant fermentation pro starch unit (SSU) and is based on the degree of hydrolysis
cess is an alcohol product, preferably ethanol. The end prod- 15 of soluble potato starch substrate (4% DS) by an aliquot of
uct produced according to the process may be separated and/ the enzyme sample at pH 4.5, 50° C. The reducing sugar
or purified from the fermentation media. Methods for content is measured using the DNS method as described in
separation and purification are known, for example by sub Miller, G.
jecting the media to extraction, distillation and column chro L. (1959) Anal. Chem. 31:426-428. One unit of the enzyme
matography. In some embodiments, the end product is iden- 20 activity (SSU) is equivalent to the reducing power of 1 mg
tified directly by submitting the media to high-pressure liquid of glucose released per minute at the specific incubation
chromatography (HPLC) analysis. condi tions.
In further embodiments, the mash may be separated by for Glucoamylase activity was measured using a well-known
example centrifugation into the liquid phase and solids phase assay which is based on the ability of glucoamylase to cata
and end products such as alcohol and solids recovered. The 25 lyze the hydrolysis of p-nitrophenyl-alpha-D-glucopyrano
alcohol may be recovered by means such as distillation and side (PNPG) to glucose and p-nitrophenol. At an alkaline
molecular sieve dehydration or ultra filtration. pH, the nitrophenol; forms a yellow color that is
In some embodiments, the yield of ethanol will be greater proportional to glucoamylase activity and is monitored at
than 8%, 10%, 12%, 14%, 16% and 18% by volume. The 400 nm and com- pared against an enzyme standard
ethanol obtained according to process of the invention may be measured as a GAU.
30 used as a fuel ethanol, potable ethanol or industrial ethanol. One "GlucoamylaseActivity Unit" (GAU) is the amount
In further embodiments, the end product may include the of enzyme that will produce 1 gm of reducing sugar,
fermentation co-products such as distillers dried grains calculated as glucose per hour from a soluble starch
(DDG) and distiller's dried grain plus solubles (DDGS), substrate (4% DS) at pH 4.2 and 60° C.
Brix, the measurement of total solubilized solid content at
a given temperature was determined by measurement with a
Refractometer.
which may be used as an 35 Determination of total
animal feed. starch content: The enzyme-
enzyme
EXPERI starch liquefaction and
MENTA saccharification process
L was used to determine the
total starch content. In a
The following examples typical analysis, 2 g of
are provided in order to dry sample was taken in a
demon strate and further 100 ml Kohlraucsh flask
illustrate certain preferred and 45 ml of MOPS
embodiments and aspects of buffer, pH 7.0 was
the present invention and added. The slurry was
are not to be con strued as well
limiting the scope thereof. 40 stirred for 30 min.
Indeed, it is contemplated SPEZYME FRED (1:50
that these teachings will diluted in water)
find use in further (Genencor), 1.0 ml was
optimizing the process added and heated to
systems described herein. boiling for 3-5 min. The
flask was placed in an
autoclave maintained at
121°
C. for 15 min. After
autoclaving the flask was
placed in a water bath at
95° C. and 1 ml of 1:50
diluted SPEZYME
In the disclosure and Centigrade); H20 (water);
experimental section which dH 20 (deionized water);
follows, 45 the following dIH20 (deionized water,
abbreviations apply: GA Milli-Q filtration); g or gm
(glucoamylase); wt% (weight (grams); µg (micrograms);
percent); ° C. (degrees mg (milli grams); kg
 US 7,968,318 B2
13 14
(kilograms); µL (microliters); FRED was added and
ml and mL (milli- 50 incubated for 45 min. The
liters); mm (millimeters); µm pH was adjusted to pH 4.2
(micrometer); M (molar); mM and the temperature was
(millimolar); µM reduced to 60° C. This was
(micromolar); U (units); MW followed by addition of20 ml
(molecular weight); sec acetate buffer, pH 4.2.
(seconds); min(s) Saccharification was carried
(minute/minutes); hr(s) (hour/ out by adding 1.0 ml of
hours); DO (dissolved 1:100 diluted OPTIDEX L-
oxygen); WNV (weight to 400 (Genencor) and the
volume); W/W (weight to incubation was continued for
weight); VN (volume to 18 hr at 60° C. The enzyme
volume); Genencor 55 reaction was ter minated by
(Genencor International, Inc., heating at 95° C. for 10 min.
Palo Alto, Calif.); MT (Metric The total sugar composition
ton); and ETOH (ethanol). was determined by HPLC
The following enzyme analysis using glu cose as a
preparations were used in standard. The soluble starch
the examples below: hydrolysate from water
SPEZYME Ethyl (available extraction of a sample at
from Genencor)-a bacterial 60 room temperature without
alpha-amylase obtained from a enzy matic treatment was
genetically modified strain of subtracted from the total
Bacillus licheniformis. sugar.
GCl00-an experimental Determination of the%
bacterial alpha-amylase dis solubilized solids-a 7 ml
closed in US 2006/0014265. sample was placed in a
Humicola grisea small screw cap test tube
glucoamylase (HGA) having (pH adjusted to 5.0 to 6.0)
the amino 65 acid sequence and 0.007 ml SPEZYME
disclosed as SEQ ID NO: 3 of Fred was added to the tube.
WO 2005/ 052148. The test tube was placed in
a boiling water bath for 10
min and gently mixed at
various times during the
incubation. After 10 min
the tube was removed and
placed in a 80° C. water
bath for 1 hr. The tube was
cooled and centrifuges. The
Brix of the supernatant was
determined and compared
to a control sample. The%
solubilized solids=control
Brixxl00/sample Brix.
Ethanol and carbohydrate
determinations of the
samples were determined
using the HPLC method as
follows:
 US 7,968,318 B2
15 16
a 1.5 mL Eppendorf centrifuge tube was filled with TABLE I-continued
fer mentor mash and cooled on ice for 10 min; the test tube and allowed to 5
sample tube was centrifuged for 1 min in an containing
Eppendorftable top centri fuge; a 0.5 mL sample of the 0.05 mL of
supernatant was transferred to a 1.lN H2 SO4
stand for 5 min; degree of
5.0 mL of water polymerization
was added to the greater than 3).
test tube and
then the sample
was filtered into
a HPLC vial
through 0.2 µm
Nylon Syringe
Filter; and run
on HPLC. The
HPLC condi
tions included:
Ethanol System:
Column:
Phenomenex
Rezex Organic 10
Acid Colunm
(RHM-
Monosaccharide
) #OOH 0132-
KO (Equivalent
to Bio-Rad
87H); Colunm
Temperature:
60° C.; Mobile
Phase: 0.01 N
H2 SO4 ; Flow
Rate: 0.6
mL/min; Detec
tor: RI; and
Injection
Volume: 20 µL.
Carbohydrate
System: Colunm:
Phenomenex
Rezex Car- 15
bohydrate (RCM-
Monosaccharide)
#00H-0130-KO
(Equivalent to
20 Bio-RadTABLE2 87H);
Colunm
-----------
STARGEN %VIV
Temperature: 70°
001 ETOH %V/VETOH %V/VETOH
GAU/g
C.;
24hrs
Mobile Phase:
48 hrs 72 hrs
Nanopure DI H2O;
25 0.1 Flow
10.11 Rate: 11.740.8 13.64
0.2 10.36 12.19 14.62
0.4
mL/min;
10.83
Detector:
12.73 15.35
RI; Injection
Volume: 10 µL
(3% DS material).
The colunm
separated based on
the molecular
weight of the
saccharides, which
are designated as
DPl (glucose);
DP2
(disaccharides);
DP3
(trisaccharides)
and DP>3
(oligosac charide
sugars having a
 % Starch
US 7,968,318 B2 Solu- % % %
Grain Time BRIX bilized Glucose DP2 DP3 DP>3
15 16
com (32% DS), yeast Refined 2 14.4 a 1.40 10.37 15.27 72.96
fermentations were Cornstarch r
conducted at pH 4.2, 32° 4 16.8 2.34 11.60 15.22 70.84
C. in the presence of 400 6 17.8 S 5.14 12.27 15.13 67.45
12 19.1 t 4.94 13.21 15.62 66.22
ppm urea; Red Star Red
24 20.9 60.0
a 5.79 14.40 17.42 62.39
yeast (Fermentus);
r
STARGEN 001; and 0.1
c
After 24 hours, using the SAPU/g DS protease in a
h
samples from whole ground 125 ml flask. HPLC data
are illustrated in Table 2. o
f
E
X W
A h
M o
P l
L e
E
G
1 r
o
S u
o n
l d
u
b C
i o
l r
i n
z
a a
t n
i d
o Fractionated Com
n
30
a
n
EXAMPLE2
d
This experiment was run on three
E different com granular
t starch substrates was maintained at
h (A) 370 g of whole 60° C. During the
a ground corn having incubation the
n a moisture content slurry was gently
o of 13.3%; (B) stirred with an
l 354.2 g com overhead mixer.
endosperm hav ing After time
P a moisture content internals of
r 2, 4, 6, 12 and 24
of 9.2%; and (C) hours, the BRIX,
o refined com starch
d % solubilized
35 obtained from starch and
u having a moisture
c content of 11.8%.
t Each substrate was
i weighed and
o transferred to a
n stainless steel
vessel to make a
F final 1000 g slurry
r with water
o corresponding
m to 32% DS.
The pH of the
G slurry was adjusted
r to pH 5.5 using
a 6N 40 H2 SO4 .
n GCl00 (4.0AAU/g
u DS) was added.
l The temperature
 US 7,968,318 B2
15 16
Solubilization and transferred to a stainless ar Yeast, pH
Ethanol steel vessel containing 340 4.2, 32° C. in
Producti g water. The pH of the the presence of
on From slurry was adjusted to pH 400 ppm urea;
Milo 5.5 using 6 N sulphuric STARGEN 001
Granula acid. SPEZYME Ethyl (1.0 and 0.05
r Starch AAU/g DS) was added. SAPU/g DS) in
The temperature was a 125 ml flask.
Two pretreatments were maintained at 62° C. HPLC results
run: (A) 160 g of whole and32% DS. (B) HGA was are illustrated in
ground milo having a included in the pretreat Table 4.
moisture content of 11.6% ment as described above in
and a total starch content of (A) at the equivalent of0.1
53.3% was weighed and GAU
sugar compositions (% 45 HGA/g DS. The %
W/W) were determined, solubilized solids and %
and the results are glucose are presented in
illustrated in Table I.At 24 Table 3.
hours, 79.1%, 71.1% and
60.0% of the granular T
starch from whole ground A
com, endosperm and B
refined sugar was L
solubilized during the con E
tacting step respectively. 3
The% glucose of the
hydrolyzate at
24 hours was 65.22% for whole ground corn, 49.64% for Time %DP! % % solublized
50 Enzyme (hrs) (glucose) DP2 DP3 %DP>3 starch
endosperm and only 5.79% for refined starch.
SPEZYME 2 57.71 24.60 10.42 7.27
Ethyl
TABLE 1 4 64.41 22.50 9.13 3.95
6 67.16 21.83 8.16 2.85
% Starch 55 24 86.50 9.91 2.95 0.63 54.7
Solu- % % % SPEZYME 2 84.34 9.48 1.47 4.72
Grain Time BRIX bilized Glucose DP2 DP3 DP> 3 Ethyl+HGA
4 87.72 8.07 1.18 3.03
Whole 2 11.5 31.60 21.36 13.48 33.56 6 88.91 7.68 1.03 2.37
24 93.10 5.26 0.59 1.05 69.3
4 14.0 37.56 24.31 13.65 24.48
66 16.3 41.33 25.42 13.36 19.88
120 18.2 46.23 26.18 12.63 14.96
c
o 24 20.0 79.1 65.22 22.50 7.36 4.92
Endosperm 2 13.4 27.12 13.85 8.88 50.15
4 16.1 33.01 16.45 9.39 41.15
6 17.5 36.56 18.18 9.59 35.67
The feedstock (mash) from the HGA pretreatment
12 21.7 47.43 20.98 9.65 21.94
described above24was 22.9
evaluated under
71.1 regular21.33
49.64 yeast 9.52
fermenta-
19.51
65
ti
o
n
c
o
n
d
it
i
o
n
s
(
e
.
g
.
R
e
d
S
t
 US 7,968,318 B2
17 18
TABLE4 adjusted to pH 5.5. The Brix was measured at 2, 4 6, and 24
hrs. The% soluble starch and% glucose were determined
%VIV and the results are illustrated in Table 7.
GAU/g Ethanol % VIV Ethanol
Pre-treatment STARGEN 001 24 hrs 48 hrs
TABLE 7
SPEZYME 0.1 10.02 11.98
Ethyl+ HGA % %WN
0.2 10.22 12.75 solubilized DP! % WIV % WIV % WIV
Time hrs Brix starch (glucose) DP2 DP3 .DP3

10 11 39.2 27.5 19.8 12.7 39.6

2 12.5 44.6 31.2 22.2 13.4 32.6
EXAMPLE3 4 14.8 52.8 33.4 24.0 13.8 28.7
Effect6of Temperature
16.2 on
57.5 Glucose
33.8Production
24.4 From
13.9 27.7 24 16.4 58.4
Whole Ground Milo
15
After 24 hours, yeast
Incubation of a 30% ds
fermentations were conducted
aqueous slurry of whole at pH 4.2, 30° C. in
ground milo at pH 5.5 thepresenceof0.75 GAU/gDS
containing GCl00 STARGEN00l, 400 ppm urea,
(4.0AAU/gDS)wascarried and Angel yeast (Jiangxi,
out at 60° C., 65° C. and China) at 0.4%. HPLC
70° C. After 6 hours of the samples were taken at 24, 48
incuba tion, the samples and 67 hrs (Table 8).
were withdrawn and
centrifuged to separate
the insolubles. The Brix of the clear supernatant was mea- 20

sured. The oligosaccharide composition (DP2, DP3, and

DP>3) of the clear supernatant was determined by HPLC. Time
The% solubilized starch and% glucose were determined and hrs

the results are illustrated in Table 5. 24

25 48
67
TABLES
% %W/W
Solubilized DP!
%W/W We claim:
%W/W 1. A method of producing
%W/Wglucose from a granular starch
"C. Starch (Glucose)
DP3 DP >3
substrate comprising:
30
a) contacting a slurry
60 69.9 52.48 comprising granular
12.08 8.36
starch obtained from a
65 68.2 52.61
10.41 13.56 plant material with an a-
70 61.9 36.49 amylase at a temperature
10.27 34.26 below the starch
gelatinizationtemperature
As the incubation temperature of the granu lar starch to
increased from 60° C. to 70° produce oligosaccharides
35 and allowing endogenous
C. during the contacting step plant starch hydrolyzing
with whole ground milo, the enzymes to hydro- lyze
solubilization of starch and the said oligosaccharides,
glucose content decreased. and
This suggests that the alpha- b) producing a mash
amylase may be inactivated at comprising at least 10%
70° C. More than 50% of the glucose, wherein the
solubilized starch was hydro contacting step is
lyzed to glucose at 65° C. conducted for a period of
suggesting the endogenous 5 minutes to 48 hours at a
plant 40 starch hydrolyzing pH of3.5 to 7.0.
enzymes are capable of 2. The method according to
hydrolyzing the soluble claim 1, wherein the a-amy
oligosaccharides into glucose. lase is derived from a Bacillus
The feedstock from the stearothermophilus,a Bacillus
pretreatment described above licheniformis or a Bacillus
at 65° C. was evaluated under amyloliquefaciens.
3. The method according to
regular fermentation
conditions claim 1, wherein the
amount of
as essentially described in was 30.The results are illustrated
example 2; except the % DS in Table 6.
 US 7,968,318 B2
17 18
45 a-amylase P
supplied in the bilizing granular starch to
contacting step is r m produce oligosaccharides
between 0.01 to o ak and allowing endogenous
10.0 a-amylase d ea plant starch hydrolyzing
units per gram u 25 enzymes to hydrolyze said
dry solids c % oligosaccharides, wherein
content (AAU/g t D said con tacting is at a pH
DS). i S of3 .5 to 7.0; at a
4. The method o slu temperature below the starch
according to n rry gelatinization temperature of
claim 1, . the granular starch; and for a
wherein the F G period of 5 minutes to 24
tempera r Cl hours,
o 00 b) obtaining a mash
ture is between 50° C. and 70° C. m (4. comprising greater than
STARGEN 50 5. The 0 20% glucose, and
method R A
ETOH according to i A
claim 1, c U/
%VIVET wherein the e g
OH mash D
(GAU/gDS)24 comprises at 6
hrs 0 S)
72 hrs least 30% wa Starch
Granular
glucose. s
0.1 6. The
11.00 R ad
method de
ice
0.2 according to d
grai
11.07 claim 1, to
0.4 n
wherein the th
11.47 (116
plant e
g)
material is slu
havi
corn, milo, rry
EXAMPLE ng a
barley, .
4 wheat, rice star
ch Th
or e
combination cont
ent te
s thereof.
of m
55 7. The
81.5 pe
method
%; a rat
according to
moi ur
claim 6,
stur e
wherein the
plant e wa
material is cont s
fractionated ent m
com. of ai
8. A process 14% nt
for and ai
producin a ne
g ethanol parti d
comprisi cle at
ng: size 65
a) contact that °
ing a pass C.
slurry es an
compri thro d
sing ugh p
granula a 30 H
r starch mes
obtaine h
d from scre
plant en
materia was
l with mix
an a- ed
amylas with
e 284
capable g of
of wat
solu er to
Solubilization and Ethanol
 US 7,968,318 B2
19 20
c) fermenting said mash in the presence of a fermenting 18. The process according to claim 8, wherein the slurry
microorganism and starch hydrolyzing enzymes at a has between 5-60% DS granular starch.
temperature of between 10° C. and 40° C. for a period 19. The process of claim 8 further comprising contacting
of 10 hours to 250 hours to produce ethanol. the mash with an aqueous solution comprising backset to
5 dilute the% DS prior to the fermentation step.
9. The process according to claim 8 further comprising
20. The process according to claim 8, wherein the
recovering the ethanol.
granular starch is obtained from com, milo, barley, wheat,
10. The process according to claim 8, wherein the a-
rice or combinations thereof.
amy lase is a bacterial a-amylase. 21. The process according to claim 12, wherein the tem-
11. The process according to claim 10, wherein the a- 10 perature is between 60° C. and 70° C.
amy lase is derived from a Bacillus stearothermophilus,a 22. The process according to claim 15, wherein said mash
Bacillus licheniformis or a Bacillus amyloliquefaciens. comprises greater than 40% glucose after contacting
12. The process according to claim 8, wherein the between 15 minutes and 6 hours.
contact ing step is conducted at a temperature of between 23. The process according to claim 17, wherein the addi-
50° C. and 70° C. 15 tional enzymes are selected from the group of glucoamylases,
13. The process according to claim 8, wherein the phytases and proteases.
contact ing step is conducted at a pH of between 5.0 and 24. The process according to claim 18, wherein the% DS
6.0. granular starch is between 20-40% DS.
14. The process according to claim 8, wherein the 25. The process according to claim 20, wherein the %
contact ing is for a period of 15 minutes to 12 hours. 20 glucose after 6 hours is 30%.
15. The process according to claim 8, wherein said mash 26. The process according to claim 20, wherein the %
comprises greater than 30% glucose after contacting glucose after 6 hours is 40%.
between 15 minutes and 6 hours. 27. The process according to claim 23, wherein the addi
16. The process according to claim 8, further comprising tional enzyme is a phytase.
clarifying said mash by centrifugation before the fermenting 25 28. The process according to claim 23, wherein the addi
tional enzyme is a protease.
step.
17. The process according to claim 8, further comprising
* * * * *
adding additional enzymes to the contacting step.