U.S. patent application number 13/289026 was filed with the patent office on 2014-01-09 for fermentation processes.
This patent application is currently assigned to NOVOZYMES A/S. The applicant listed for this patent is Randy Deinhammer, Rikke Monica Festersen. Invention is credited to Randy Deinhammer, Rikke Monica Festersen.
Application Number | 20140011250 13/289026 |
Document ID | / |
Family ID | 38523304 |
Filed Date | 2014-01-09 |
United States Patent
Application |
20140011250 |
Kind Code |
A1 |
Deinhammer; Randy ; et
al. |
January 9, 2014 |
Fermentation Processes
Abstract
The present invention provides a fermentation process for
producing a fermentation product from starch-containing material
wherein one or more antibacterial agents are added before and/or
during fermentation.
Inventors: |
Deinhammer; Randy; (Wake
Forest, NC) ; Festersen; Rikke Monica; (Herlev,
DK) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Deinhammer; Randy
Festersen; Rikke Monica |
Wake Forest
Herlev |
NC |
US
DK |
|
|
Assignee: |
NOVOZYMES A/S
Bagsvaerd
DK
|
Family ID: |
38523304 |
Appl. No.: |
13/289026 |
Filed: |
November 4, 2011 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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12282732 |
Sep 12, 2008 |
8076112 |
|
|
PCT/US2007/064592 |
Mar 22, 2007 |
|
|
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13289026 |
|
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60784777 |
Mar 22, 2006 |
|
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Current U.S.
Class: |
435/162 ; 426/13;
426/48 |
Current CPC
Class: |
Y02E 50/17 20130101;
C12P 19/14 20130101; Y02E 50/10 20130101; C12C 11/00 20130101; C12P
7/06 20130101; C12P 7/14 20130101 |
Class at
Publication: |
435/162 ; 426/48;
426/13 |
International
Class: |
C12C 11/00 20060101
C12C011/00; C12P 7/14 20060101 C12P007/14 |
Claims
1-28. (canceled)
29. A process for producing a fermentation product, comprising: (a)
liquefying a starch-containing material to form a liquefied starch;
(b) saccharifying the liquefied starch using a carbohydrate source
generating enzyme to form a sugar; and (c) fermenting the sugar
using a fermenting organism under conditions suitable to produce
the fermentation product, wherein one or more lysozymes are added
before or during fermentation and wherein said one or more
lysozymes are added at a concentration sufficient to inhibit growth
of contaminating lactic acid bacterial cells.
30. The process of claim 29, wherein steps (b) and (c) are carried
out sequentially or simultaneously.
31. The process of claim 29, wherein a slurry of the
starch-containing material is heated to above the gelatinization
temperature of the starch-containing material.
32. The process of claim 29, wherein the one or more lysozymes are
added during liquefaction.
33. The process of claim 29, wherein the one or more lysozymes are
added during saccharification.
34. The process of claim 29, wherein the one or more lysozymes are
added during fermentation.
35. The process of claim 29, wherein the fermentation product is an
alcohol.
36. The process of claim 35, wherein the alcohol is ethanol.
37. The process of claim 29, wherein the starch-containing starting
material is whole grains, whole corn, or wheat grains.
38. The process of claim 29, wherein the liquefaction is carried
out using an alpha-amylase.
39. The process of claim 29, wherein the bacterial cells are
gram-positive bacteria or gram-negative bacteria cells.
40. The process of claim 39, wherein the bacterial cells are
Lactobacillus cells.
41. A process for producing a fermentation product, comprising: (a)
saccharifying a starch-containing material at a temperature below
the initial gelatinization temperature of the starch-containing
material to form a sugar; and (b) fermenting the sugar using a
fermenting organism under conditions suitable to produce the
fermentation product; wherein one or more lysozymes are added
before or during fermentation and wherein said one or more
lysozymes are added at a concentration sufficient to inhibit growth
of contaminating lactic acid bacterial cells.
42. The process of claim 41, wherein the saccharification and
fermentation are carried out sequentially or simultaneously.
43. The process of claim 41, wherein the fermentation is carried
out at a temperature in the range from 20-40.degree. C.
44. The process of claim 41, wherein the one or more lysozymes are
added during saccharification.
45. The process of claim 41, wherein the bacterial cells are
gram-positive bacteria or gram-negative bacteria cells.
46. The process of claim 45, wherein the bacterial cells are
Lactobacillus cells.
47. The process of claim 41, wherein the starch-containing material
is granular starch.
48. The process of claim 41, wherein the fermentation product is an
alcohol.
49. The process of claim 48, wherein the alcohol is ethanol.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 12/282,732 filed on Sep. 12, 2008, now allowed, which is a 35
U.S.C. 371 national application of PCT/US2007/064592 filed on Mar.
22, 2007, which claims priority or the benefit under 35 U.S.C. 119
of US provisional application No. 60/784,777 filed on Mar. 22,
2006, the contents of which are fully incorporated herein by
reference.
FIELD OF THE INVENTION
[0002] The present invention relates to processes for producing
fermentation products from starch-containing material, including
processes for producing ethanol.
BACKGROUND OF THE INVENTION
[0003] Fermentation processes are used for making a vast number of
commercial products, including alcohols (e.g., ethanol, methanol,
butanol, 1,3-propanediol); organic acids (e.g., citric acid, acetic
acid, itaconic acid, gluconic acid, gluconate, lactic acid,
succinic acid, 2,5-diketo-D-gluconic acid); ketones (e.g.,
acetone); amino acids (e.g., glutamic acid); gases (e.g., H.sub.2
and CO.sub.2), and more complex compounds, including, for example,
antibiotics (e.g., penicillin and tetracycline); enzymes; vitamins
(e.g., riboflavin, B.sub.12, beta-carotene); hormones, and other
compounds which are difficult to produce synthetically.
Fermentation processes are also commonly used in dairy (e.g., in
the production of yogurt and cheese), leather, and tobacco
industries.
[0004] Fermentation products are produced industrially in large
fermentation tanks capable of holding upwards of 10 cubic meters
fermentation medium. In order to build up a suitable fermenting
organism population and concentration in the fermentation tank a
fermentation process usually requires a process time of between 48
and 120 hours or more. Because of the large-sized tanks and long
fermentation times it is difficult to maintain the fermentation
system free of contamination. Unwanted contaminant bacteria are
often gram-positive bacteria from the genus Lactobacillus that
converts glucose into lactic acid and acetic acid. Also
gram-negative bacteria are known to contaminate fermentation
processes. Unfortunately the fermentation conditions are usually
conducive for bacterial growth. If bacterial contamination occurs
the entire fermentation tank must be emptied, cleaned and
sterilized and the fermentation medium is useless. This is of
course time-consuming and costly. Further, many bacteria compete
with the fermenting organism for the sugar. This results in a
reduced fermentation yield.
[0005] Bayrock et al., 2003, Appln. Microbiol. Biotechnol.
62:498-502 disclose control of Lactobacillus contaminants in
continuous fuel ethanol fermentations by constant or pulse addition
of penicillin G.
[0006] Today antibiotics, heat and chemical disinfectants are used
for killing and/or inhibiting growth of unwanted bacteria. These
disinfectants are added to the fermentation before or during
fermentation. The known antibacterial agents including antibiotics,
such as penicillin, are sometimes not desired. Therefore, there is
a need for further means for killing and/or inhibiting unwanted
bacteria growth during fermentation processes.
SUMMARY OF THE INVENTION
[0007] The object of the present invention is to provide
fermentation processes, or processes including a fermentation step,
wherein unwanted bacteria are killed and/or unwanted bacteria
growth is inhibited.
[0008] In the first aspect the present invention provides processes
for producing a fermentation product from starch-containing
material using a fermenting organism, wherein one or more
antibacterial agents are added before and/or during
fermentation.
[0009] In the second aspect the invention relates to a process for
producing a fermentation product from starch-containing material
comprising the steps of:
[0010] (a) liquefying starch-containing material;
[0011] (b) saccharifying using a carbohydrate source generating
enzyme;
[0012] (c) fermenting using a fermenting organism, wherein one or
more antibacterial agents are added before and/or during
fermentation.
[0013] In the third aspect the invention relates to a process for
producing a fermentation product from starch-containing material
comprising:
[0014] (a) saccharifying starch-containing material at a
temperature below the initial gelatinization temperature of said
starch-containing material,
[0015] (b) fermenting using a fermenting organism, wherein one or
more antibacterial agents are added before and/or during
fermentation.
[0016] Finally the invention also relates to use of antibacterial
agents for killing and/or inhibiting bacterial growth in
fermentation product production processes.
Definitions
[0017] The term "antibacterial activity" means activity which is
capable of killing and/or inhibiting growth of bacteria. An
"antibacterial peptide" is a peptide capable of killing and/or
inhibiting growth of bacteria. In a similar manner an
"antibacterial polypeptide" and "antibacterial enzyme: are
polypeptides and enzymes, respectively, capable of killing and/or
inhibiting growth of bacteria. In the context of the present
invention the term "inhibiting growth of microbial bacteria" is
intended to mean that the bacteria are in the non-growing state,
i.e., that they are not able to multiplicate. It is to be
understood that an "antibacterial peptide" or the like may also be
capable of killing and/or inhibiting growth of other microbial
cells, such as certain fungal cells.
[0018] When used herein, a "fragment" of an amino acid sequence,
peptide, polypeptide, enzyme etc. means a subsequence wherein one
or more amino acids have been deleted from the amino and/or
carboxyl terminus. Preferably one or more amino acids have been
deleted from the carboxyl terminus. A fragment should also have
antibacterial activity.
[0019] An antimicrobial peptide, polypeptide, protein, enzyme or
the like used in a process of the invention may be a "variant"
which comprises, preferably consists of, an amino acid sequence
that has at least one substitution, deletion and/or insertion of an
amino acid as compared to the parent/wild-type amino acid sequence.
Such variant may be constructed by any technique known in the art,
such as by site-directed/random mutagenesis and domain shuffling
techniques. In one embodiment the amino acid change(s) (in the
variant as well as in parent/wild-type sequence) is(are) of minor
nature, such as conservative amino acid substitution(s), that do
not significantly affect the folding and/or activity of the
molecule.
[0020] The term "homology" between two amino acid sequences or
between two nucleotide sequences is described by the parameter
"identity".
[0021] The degree of "identity" between two amino acid sequences is
determined by using the program FASTA included in version
2.0.times. of the FASTA program package (see Pearson and Lipman,
1988, "Improved Tools for Biological Sequence Analysis", PNAS
85:2444-2448; and Pearson, 1990, "Rapid and Sensitive Sequence
Comparison with FASTP and FASTA", Methods in Enzymology 183:63-98).
The scoring matrix used was BLOSUM50, gap penalty was -12, and gap
extension penalty was -2.
[0022] The degree of identity between two nucleotide sequences is
determined using the same algorithm and software package as
described above. The scoring matrix used was the identity matrix,
gap penalty was -16, and gap extension penalty was -4.
[0023] The term "unwanted bacteria" means in context of the
invention bacteria that is undesired in that they may impact
fermentation product production in a negative way, for instance, by
converting the fermenting organism's substrate into an undesired
fermentation product. An example of unwanted contaminant bacteria
in, e.g., alcohol production, including ethanol production, is
Lactobacillus that converts glucose into lactic acid and acetic
acid.
BRIEF DESCRIPTION OF THE DRAWING
[0024] FIG. 1 shows the growth of mixture of four Lactobacillus
strains over 24 hours at different concentrations of antibacterial
Peptide A.
[0025] FIG. 2 shows the growth of mixture of four Lactobacillus
strains over 24 hours at different concentrations of antibacterial
Peptide B.
[0026] FIG. 3 shows the total count of Lactobacilli after 0, 24,
48, and 72 hours of SSF.
[0027] FIG. 4 shows the antibacterial effect of 0-1,000 mg
Lysozyme/L fermentation medium on Lactobacillus over 48 hours.
DESCRIPTION OF THE INVENTION
[0028] The object of the present invention is to provide
fermentation processes or processes including a fermentation step
wherein unwanted bacteria are killed and/or unwanted bacteria
growth is inhibited. Unwanted bacteria themselves and their
metabolic end-products, such as lactic acid and/or acetic acid,
lead to reduced fermentation yields which lead to considerable
economical loss to the producer (see Thomas et al., 2001, J.
Applied Microbiology 90: 819-828). The unwanted bacteria compete
with the fermenting organism (e.g., yeast) for sugar (carbon
source) in the fermentation medium. The lactic acid and/or acetic
acid produced by the unwanted bacteria may also have a negative
impact on yeast growth.
[0029] The present inventors have found that antibacterial agents
may advantageously be used to kill and/or inhibit growth of
unwanted bacteria which are known to contaminate fermentation
processes. A process of the invention may be used as an alternative
to, e.g., adding antibiotics, such as especially penicillin, to
fermentation processes, which may be undesired for one reason or
another. A process of the invention may result in a fermentation
yield that is increased compared to the yield obtained in a
corresponding process where no antibacterial agent is added.
[0030] According to the invention especially bacterial
contamination by lactic acid and/or acetic acid producing bacteria
may be prevented and/or reduced. Lactic acid and/or acetic acid
producing bacteria of especially the genus Lactobacillus are known
to contaminate fermentation processes. Examples of species of
Lactobacillus that has been found to contaminate fermentation
processes include strains of Lactobacillus collinoides,
Lactobacillus brevis, Lactobacillus fermentum, Lactobacillus
paracasei, Lactobacillus plantarum, and/or Lactobacillus rhamnosus,
or a mixture thereof.
Processes of the Invention
[0031] In the first aspect the invention relates to processes for
producing a fermentation product from starch-containing material
using a fermenting organism, wherein one or more antibacterial
agents are added before and/or during fermentation.
[0032] A fermentation process of the invention includes, without
limitation, fermentation processes used to produce fermentation
products including alcohols (e.g., ethanol, methanol, butanol,
1,3-propanediol); organic acids (e.g., citric acid, acetic acid,
itaconic acid, gluconic acid, gluconate, lactic acid, succinic
acid, 2,5 diketo-D-gluconic acid); ketones (e.g., acetone); amino
acids (e.g., glutamic acid); gases (e.g., H.sub.2 and CO.sub.2),
and more complex compounds, including, for example, antibiotics
(e.g., penicillin and tetracycline); enzymes; vitamins (e.g.,
riboflavin, B.sub.12, beta-carotene); hormones, and other
compounds. Fermentation processes also include fermentation
processes used in dairy industry (e.g., fermented dairy products),
leather industry and tobacco industry. Preferred fermentation
processes include alcohol fermentation processes, which are well
known in the art. Preferred fermentation processes are anaerobic
fermentation processes. In an embodiment the fermentation process
of the invention is part of a process further comprises a
liquefaction step and/or a saccharification step. In a preferred
embodiment the fermentation process is a step in a simultaneous
saccharification and fermentation process (SSF process) or a
one-step fermentation process of uncooked starch-containing
material (sometimes referred to as simultaneous liquefaction,
saccharification and fermentation (LSF)). Examples of one-step
processes include the processes disclosed in U.S. Pat. No.
4,316,956; US 2004/0234649 and WO 2003/066816 and WO 2003.066826
(which references are all incorporated by reference).
[0033] The fermentation process of the invention may in one
embodiment be carried out at a temperature in the range from
20-40.degree. C., preferably 30-35.degree. C., especially around
32.degree. C. This is usually the case when producing alcoholic
fermentation products such as ethanol, especially fuel ethanol,
potable ethanol and/or industrial ethanol.
[0034] The pH during fermentation may in a preferred embodiment be
in the range between 4-7, preferably in the range between 5 and
6.
[0035] In one embodiment the antibacterial agent is added during
liquefaction and/or saccharification, i.e., before fermentation, or
during simultaneous saccharification and fermentation (SSF). In
another embodiment the antibacterial agent is added to backset
and/or thin stillage recycled to typically the liquefaction and/or
fermentation steps.
[0036] Fermentation processes are usually carried out as batch
fermentation, i.e., fermentation conducted from start to finish in
a single tank, or continuous fermentation, i.e., a steady state
fermentation system that operates without interruption and where
each stage of fermentation occurs in a separate section of the
fermentation system, and flow rates are set to correspond to
required residence times. In other words, the individual process
steps in a process comprising a fermentation process of the
invention may be performed batch wise or continuously. Processes
where all process steps are performed batch wise, or processes
where all process steps are performed continuously, or processes
where one or more process steps are performed batch wise and one or
more process steps are performed continuously are contemplated
according to the invention. The cascade process is an example of a
process where one or more process steps are performed continuously
and as such contemplated for the invention. For further information
on the cascade process and other especially ethanol processes
consult "The Alcohol Textbook", Ethanol production by fermentation
and distillation. Eds. T. P. Lyons, D. R. Kesall and J. E. Murtagh.
Nottingham University Press 1995. In a preferred embodiment the
fermentation process of the invention is part of a continuous
fermentation product production process.
[0037] In a preferred embodiment the antibacterial agent is left in
contact with the fermentation medium for between 1 minute and 48
hours, preferably at least 1 hour, especially at least 24 hours
before inoculation of the fermenting organism.
[0038] A process of the invention may be carried out for a period
of 1 to 250 hours, preferably from 25 to 190 hours, more preferably
from 30 to 180 hours, more preferably from 40 to 170 hours, even
more preferably from 50 to 160 hours, yet more preferably from 60
to 150 hours, even yet more preferably from 70 to 140 hours, and
most preferably from 80 to 130 hours.
Fermentation Medium
[0039] "Fermentation media", "fermentation medium" or "fermentation
broth" refers to the environment in which fermentation is carried
out and which includes the fermentation substrate, that is, the
carbohydrate source that is metabolized by the fermenting organism.
The fermentation medium, including fermentation substrate and other
raw materials used in the fermentation process of the invention may
be processed, e.g., by milling, liquefaction and/or
saccharification or other desired steps prior to or simultaneously
with the fermentation process. Accordingly, the fermentation medium
can refer to the medium before the fermenting organism is added,
such as, the medium in or resulting from liquefaction and/or
saccharification, as well as the medium which comprises the
fermenting organism, such as, the medium used in simultaneous
saccharification and fermentation processes (SSF) or one-step
fermentation (LSF) of, e.g. uncooked raw material.
Fermenting Organisms
[0040] "Fermenting organism" refers to any organism suitable for
use in a desired fermentation process. Suitable fermenting
organisms according to the invention are able to ferment, i.e.,
convert, sugars, such as glucose or maltose, directly or indirectly
into a desired fermentation product. Examples of fermenting
organisms include fungal organisms, such as especially yeast.
Preferred yeast includes strains of Saccharomyces spp., and in
particular, strains of Saccharomyces cerevisiae. Commercially
available yeast includes, e.g., ETHANOL RED.TM. yeast (available
from Fermentis/Lesaffre, USA), FALI (available from Fleischmann's
Yeast, USA), SUPERSTART.TM. and THERMOSACC.TM. fresh yeast
(available from Ethanol Technology, WI, USA), BIOFERM AFT and XR
(available from NABC--North American Bioproducts Corporation, GA,
USA), GERT STRAND (available from Gert Strand AB, Sweden), and
FERMIOL (available from DSM Specialties).
Starch-Containing Materials
[0041] Any suitable starch-containing material, including granular
starch, may be used as substrate according to the present
invention. The material is generally selected based on the desired
fermentation product. Examples of starch-containing materials
suitable for use in a process of present invention include tubers,
roots, stems, whole grains, corn, cob, wheat, barley, rye, milo,
sago, cassava, tapioca, sorghum, sweet sorghum, rice peas, beans,
or sweet potatoes, or mixtures thereof, or cereals,
sugar-containing raw materials, such as molasses, fruit materials,
sugar cane or sugar beet, potatoes, and cellulose-containing
materials, such as wood or plant residues, or mixtures thereof.
Contemplated are both waxy and non-waxy types of corn and
barley.
[0042] Suitable substrates also include carbohydrate sources, in
particular, low molecular sugars DP.sub.1-3 that can be metabolized
by the fermenting organism, and which may be supplied by direct
addition to the fermentation medium.
Antibacterial Agents
[0043] An antibacterial agent used in a process of the invention is
preferably a polymer molecule, such as a polymer molecule
consisting of an amino acid sequence. The amino acid sequence may
be a peptide, polypeptide, protein or enzyme or the like.
[0044] In a preferred embodiment the antibacterial agent is a
peptide, polypeptide, protein, enzyme or the like capable of
killing and/or inhibiting growth of unwanted bacteria, preferably
gram positive bacteria and/or gram negative bacteria, especially
gram positive bacteria of the genus Lactobacillus.
[0045] The antibacterial peptide, polypeptide, protein, enzyme or
the like may be of microbial, such as fungal or bacterial origin,
but may also be synthetically produced.
[0046] In a preferred embodiment the antibacterial agent is a
defensin or defensin-like peptide. In a preferred embodiment the
antibacterial peptide is a fungal defensin, preferably derivable
from Pseudoplectania nigrella, especially Pseudoplectania nigrella
CBS 444.97. Specifically contemplated is the peptide disclosed as
amino acids 1-40 in SEQ ID NO: 2 in WO 2003/044049 (which is hereby
incorporated by reference) or a fragment thereof having
antibacterial activity or a variant therefore having antibacterial
activity having at least 80%, preferably at least 90%, more
preferably at least 95%, even more preferably at least 97%,
especially at least 99% identity to amino acids 1-40 in SEQ ID NO:
2 in WO 2003/044049.
[0047] In a specific embodiment the antibacterial agent is the
peptide Novispirin or a Novispirin variant selected from the group
disclosed as SEQ ID NOS: 1-37 in WO 2002/00839, especially G10
disclosed as SEQ ID NO: 17 in WO 2002/00839 (which is incorporated
by reference). In a preferred embodiment the antibacterial peptide
is a variant of Novispirin G10 (SEQ ID NO: 1 in WO 2005/105831).
Contemplated variants include any of the variants disclosed as SEQ
ID NOS: 2-116 in WO 2005/105831.
[0048] In another preferred embodiment the antibacterial agent is
an antibacterial protein or enzyme such as Lysozyme. Lysozyme may
be of any origin, such as hen Lysozyme.
[0049] The antibacterial agent(s) is(are) added in concentrations
sufficient to kill and/or inhibit growth of bacteria cells,
preferably gram positive bacteria and/or gram negative bacteria
cells, especially gram positive bacteria cells of the genus
Lactobacillus, including Lactobacillus brevis, Lactobacillus
collinoides, Lactobacillus fermentum, Lactobacillus paracasei,
Lactobacillus plantarum, and/or Lactobacillus rhamnosus, or
mixtures of one or more thereof.
[0050] Other contemplated antimicrobial peptides includes:
Heliomicin (antifungal acting AMP disclosed in WO 99/53053),
Eurocin (WO 2006/050737); Piceasin, Oystrisin, Virgisin, and
Gibbosin (WO 2006/053565); Marinasin (WO 2006/097110). Other
examples of antimicrobial agents include the antifungal peptide
disclosed in WO 2002/090384. All references are hereby incorporated
in their full length.
[0051] According to the invention the antibacterial agent(s)
is(are) added in a concentration between 0.1-1000 mg/L fermentation
medium, preferably between 0.5-500 mg/L fermentation medium,
especially between 1-100 mg/L fermentation medium.
Producing Fermentation Products from Gelatinized Starch-Containing
Material
[0052] In this aspect the present invention relates to a process
for producing a fermentation product, especially ethanol, from
starch-containing material, which process includes a liquefaction
step and separately/sequentially or simultaneously performed
saccharification and fermentation steps.
[0053] Therefore, in this aspect the invention relates to a process
for producing a fermentation product from starch-containing
material comprising the steps of:
[0054] (a) liquefying starch-containing material;
[0055] (b) saccharifying using a carbohydrate-source generating
enzyme;
[0056] (c) fermenting using a fermenting organism,
wherein one or more antibacterial agents are added before and/or
during fermentation.
[0057] The fermentation product, such as especially ethanol, may
optionally be recovered after fermentation, e.g., by distillation.
The fermentation step (c) may be a fermentation process of the
invention as described herein. Suitable starch-containing starting
materials are listed in the section "Starch-Containing
Materials"-section above. In a preferred embodiment liquefaction
step (a) is performed using an alpha-amylase. Contemplated enzymes
and suitable concentrations are listed in the "Enzymes"-section
below. Fermentation is preferably carried out in the presence of
yeast, preferably a strain of Saccharomyces. Suitable fermenting
organisms are listed in the "Fermenting Organisms"-section above.
Examples of suitable antibacterial agents are listed in the section
"Antibacterial Agents" above.
[0058] The antibacterial agent may also be added during
liquefaction and/or saccharification, i.e., before initiation of
fermentation. Alternatively the antibacterial agent may be added to
the backset and/or thin stillage recycled to, e.g., the
liquefaction and/or fermentation steps. In a preferred embodiment
the antibacterial agent is left in contact with the fermentation
medium for at least between 1 minute and 48 hours, preferably at
least 1 hour, especially at least 24 hours before inoculation of
the fermenting organism.
[0059] Liquefaction is carried out by heating to above the
gelatinization temperature of the starch-containing material.
[0060] In a preferred embodiment step (b) and (c) are carried out
simultaneously (SSF process).
[0061] In a particular embodiment, the process of the invention
further comprises, prior to step (a), the steps of:
[0062] x) reducing the particle size of the starch-containing
material, preferably by milling;
[0063] y) forming a slurry comprising starch-containing material
and water.
[0064] The slurry may include water and/or process waters, such as
backset and/or thin stillage, scrubber water, evaporator condensate
or distillate, side stripper water from distillation, or other
fermentation product plant process water. In a preferred embodiment
the starch-containing material is reduced in size by either dry
milling or wet milling. However, other size reducing technologies
such as emulsifying technology, rotary pulsation may also be used.
The aqueous slurry may contain from 10-40 wt. %, preferably 25-35
wt. % starch-containing material. The slurry is heated to above the
gelatinization temperature and alpha-amylase, preferably bacterial
and/or acid fungal alpha-amylase may be added to initiate
liquefaction (thinning). The slurry may in an embodiment be
jet-cooked to further gelatinize the slurry before being subjected
to an alpha-amylase in step (a). However, it is to be understood
that liquefaction may be carried out without a jet-cooking
step.
[0065] More specifically liquefaction may be carried out as a
three-step hot slurry process. The slurry is heated to between
60-95.degree. C., preferably 80-85.degree. C., and alpha-amylase is
added to initiate liquefaction (thinning). Then the slurry may be
jet-cooked at a temperature between 95-140.degree. C., preferably
105-125.degree. C., for 1-15 minutes, preferably for 3-10 minutes,
especially around 5 minutes. The slurry is cooled to 60-95.degree.
C. and more alpha-amylase is added to finalize hydrolysis
(secondary liquefaction). The liquefaction process may be carried
out at pH 4.5-6.5, in particular at a pH between 5 and 6. Milled
and liquefied whole grains are known as mash.
[0066] The saccharification step (b) may be carried out using
conditions well known in the art. For instance, a full
saccharification process may last up to from about 24 to about 72
hours, however, it is common only to do a pre-saccharification of
typically 40-90 minutes at a temperature between 30-65.degree. C.,
typically about 60.degree. C., followed by complete
saccharification during fermentation in a simultaneous
saccharification and fermentation process (SSF process).
Saccharification is typically carried out at temperatures from
30-65.degree. C., typically around 60.degree. C., and at a pH
between 4 and 5, normally at about pH 4.5.
[0067] The most widely used process in fermentation product
production processes, especially ethanol production, is
simultaneous saccharification and fermentation (SSF), in which
there is no holding stage for the saccharification, meaning that
the fermenting organism, such as yeast, and enzyme(s) may be added
together. SSF may be carried out at a temperature in the range from
20-40.degree. C., preferably 30-35.degree. C., especially around
32.degree. C. The pH during SSF is typically in the range between 4
and 7, preferably between 5 and 6.
Producing Fermentation Products from Un-Gelatinized/Uncooked
Starch-Containing Material.
[0068] Especially in one-step fermentation processes using uncooked
starch-containing material as the starting material adding
antibacterial agents are advantageous as one-step fermentation
processes are performed at low temperatures in the range below
20-40.degree. C. For instance, one-step ethanol fermentation
processes of the invention are typically carried out at around
32.degree. C. using Saccharomyces cerevisiae as the fermenting
organism.
[0069] Therefore, in this aspect of the invention relates to a
process for producing a fermentation product from starch-containing
material comprising:
[0070] (a) saccharifying starch-containing material at a
temperature below the initial gelatinization temperature of said
starch-containing material,
[0071] (b) fermenting using a fermenting organism,
wherein one or more antibacterial agents are added before and/or
during fermentation.
[0072] The fermentation product, such as especially ethanol, may
optionally be recovered after fermentation, e.g., by distillation.
The fermentation step (b) may be a fermentation process of the
invention as described herein. Suitable starch-containing starting
materials are listed in the section "Starch-Containing
Materials"-section above. In a preferred embodiment the
starch-containing material is granular starch. Saccharification
step (a) and fermentation step (b) may be carried out sequentially
or simultaneously. In a preferred embodiment the process is carried
as a one-step fermentation process, i.e., simultaneous
saccharification and fermentation. Examples of contemplated
one-step fermentation processes, where adding of one or more
antibacterial agents in accordance with the present invention is
relevant, are described in, e.g., U.S. Pat. No. 4,316,956; WO
2003/066816, WO 2003/066826, WO 2004/081193, WO 2004/080923, WO
2005/008156, and WO2005/118795 (which are hereby incorporated by
reference). In an embodiment alpha-amylase and/or
carbohydrate-source generating enzyme(s), especially glucoamylase,
is(are) used for hydrolyzing the uncooked starch-containing
material to fermentable sugars. In a preferred embodiment the
alpha-amylase is an acid alpha-amylase, preferably an acid fungal
alpha-amylase. Contemplated enzymes and suitable concentrations are
listed in the "Enzymes"-section below. The fermentation is
preferably carried out in the presence of yeast, preferably a
strain of Saccharomyces. Suitable fermenting organisms are listed
in the "Fermenting Organisms"-section above. Examples of suitable
antibacterial agents are listed in the section "Antibacterial
Agents" above.
[0073] The antibacterial agent(s) may be added during
saccharification before initiation of fermentation. Alternatively
the antibacterial agent(s) may be added to the backset and/or thin
stillage recycled to the fermentation step. In a preferred
embodiment the antibacterial agent(s) is(are) left in contact with
the fermentation medium for at least between 1 minute and 48 hours,
preferably at least 1 hour, especially at least 24 hours before
inoculation of the fermenting organism.
[0074] According to this aspect the process of the invention is
carried out without gelatinization of the starch-containing
material. The starch-containing material remains un-cooked. In one
embodiment alpha-amylase and/or carbohydrate-source generating
enzyme(s), preferably glucoamylase, is(are) present during
saccharification and/or fermentation. According to the invention
the desired fermentation product in question, such as ethanol, can
be produced without liquefying the starch-containing material above
gelatinizing temperatures.
[0075] The term "initial gelatinization temperature" means the
lowest temperature at which gelatinization of the starch in
question is initiated. Starch heated in water in general begins
gelatinizing between 50.degree. C. and 75.degree. C.; the exact
temperature of gelatinization depends on the specific starch, and
can readily be determined by the skilled artisan. Thus, the initial
gelatinization temperature may vary according to the plant species,
to the particular variety of the plant species as well as with the
growth conditions. In the context of this invention the initial
gelatinization temperature of a given starch-containing material is
the temperature at which birefringence is lost in 5% of the starch
granules using the method described by Gorinstein and Lii, 1992,
Starch/Starke 44(12): 461-466.
[0076] Before step (a) a slurry of starch-containing material, such
as granular starch, having 20-55 wt.-% dry solids, preferably 25-40
wt. % dry solids, more preferably 30-35 wt. % dry solids of
starch-containing material may be prepared. The slurry may include
water and/or process waters, such as backset and/or thin stillage,
scrubber water, evaporator condensate or distillate, side stripper
water from distillation, or other fermentation product plant
process water. Because the process of this aspect of the invention
the invention is carried out below the gelatinization temperature
and thus no significant viscosity increase takes place, high levels
of stillage may be used if desired. In an embodiment the aqueous
slurry contains from about 1 to about 70 vol. % stillage,
preferably 15-60% vol. % stillage, especially from about 30 to 50
vol. % stillage.
[0077] The starch-containing material may be prepared by reducing
the particle size, preferably by milling, to 0.05 to 3.0 mm,
preferably 0.1-0.5 mm. After being subjected to a process of the
invention at least 85%, at least 86%, at least 87%, at least 88%,
at least 89%, at least 90%, at least 91%, at least 92%, at least
93%, at least 94%, at least 95%, at least 96%, at least 97%, at
least 98%, or preferably at least 99% of the dry solids of the
starch-containing material is converted into a soluble starch
hydrolysate.
[0078] The process according to this aspect of the invention is
conducted at temperatures below the initial gelatinization
temperature. Preferably the temperature at which step (a) is
carried out is between 30-75.degree. C., preferably between
45-60.degree. C.
[0079] In a preferred embodiment step (a) and step (b) are carried
out as a simultaneous saccharification and fermentation process. In
such preferred embodiment the process is typically carried at a
temperature between 20-40.degree. C., preferably 30-35.degree. C.,
especially around 32.degree. C. According to the invention the
temperature may be adjusted up or down during fermentation.
[0080] In an embodiment simultaneous saccharification and
fermentation is carried out so that the sugar level, such as
glucose level, is kept at a low level such as below 6 wt. %,
preferably below about 3 wt. %, preferably below about 2 wt. %,
more preferred below about 1 wt. %., even more preferred below
about 0.5 wt. %, or even more preferred 0.25% wt. %, such as below
about 0.1 wt. %. Such low levels of sugar can be accomplished by
simply employing adjusted quantities of enzyme and fermenting
organism. At such low levels of sugar catabolic repression is
avoided. A skilled person in the art can easily determine which
quantities of enzyme and fermenting organism to use. The employed
quantities of enzyme and fermenting organism may also be selected
to maintain low concentrations of maltose in the fermentation
broth. For instance, the maltose level may be kept below about 0.5
wt. % or below about 0.2 wt. %.
[0081] The process of the invention may be carried out at a pH in
the range in the range between 4-7, preferably in the range between
5 and 6.
Enzymes
Alpha-Amylases
[0082] The alpha-amylase may according to the invention be of any
origin. Preferred are alpha-amylases of fungal or bacterial
origin.
[0083] In a preferred embodiment the alpha-amylase is an acid
alpha-amylase, e.g., fungal acid alpha-amylase or bacterial acid
alpha-amylase. The term "acid alpha-amylase" means an alpha-amylase
(E.C. 3.2.1.1) which added in an effective amount has activity
optimum at a pH in the range of 3 to 7, preferably from 3.5 to 6,
or more preferably from 4-5.
Bacterial Alpha-Amylases
[0084] According to the invention a bacterial alpha-amylase may
preferably be derived from the genus Bacillus.
[0085] In a preferred embodiment the Bacillus alpha-amylase is
derived from a strain of B. licheniformis, B. amyloliquefaciens, B.
subtilis or B. stearothermophilus, but may also be derived from
other Bacillus sp. Specific examples of contemplated alpha-amylases
include the Bacillus licheniformis alpha-amylase (BLA) shown in SEQ
ID NO: 4 in WO 99/19467, the Bacillus amyloliquefaciens
alpha-amylase (BAN) shown in SEQ ID NO: 5 in WO 99/19467, and the
Bacillus stearothermophilus alpha-amylase (BSG) shown in SEQ ID NO:
3 in WO 99/19467. In an embodiment of the invention the
alpha-amylase is an enzyme having a degree of identity of at least
60%, preferably at least 70%, more preferred at least 80%, even
more preferred at least 90%, such as at least 95%, at least 96%, at
least 97%, at least 98% or at least 99% identity to any of the
sequences shown as SEQ ID NOS: 1, 2, 3, 4, or 5, respectively, in
WO 99/19467.
[0086] The Bacillus alpha-amylase may also be a variant and/or
hybrid, especially one described in any of WO 96/23873, WO
96/23874, WO 97/41213, WO 99/19467, WO 00/60059, and WO 02/10355
(all documents hereby incorporated by reference). Specifically
contemplated alpha-amylase variants are disclosed in U.S. Pat. Nos.
6,093,562, 6,297,038 and 6,187,576 (hereby incorporated by
reference) and include Bacillus stearothermophilus alpha-amylase
(BSG alpha-amylase) variants having a deletion of one or two amino
acid in position 179 to 182, preferably a double deletion disclosed
in WO 96/23873--see e.g., page 20, lines 1-10 (hereby incorporated
by reference), preferably corresponding to delta(181-182) compared
to the wild-type BSG alpha-amylase amino acid sequence set forth in
SEQ ID NO:3 disclosed in WO 99/19467 or deletion of amino acids 179
and 180 using SEQ ID NO:3 in WO 99/19467 for numbering (which
reference is hereby incorporated by reference). Even more preferred
are Bacillus alpha-amylases, especially Bacillus stearothermophilus
alpha-amylase, which have a double deletion corresponding to
delta(181-182) and further comprise a N193F substitution (also
denoted 1181*+G182*+N193F) compared to the wild-type BSG
alpha-amylase amino acid sequence set forth in SEQ ID NO:3
disclosed in WO 99/19467.
[0087] The alpha-amylase may also be a maltogenic alpha-amylase. A
"maltogenic alpha-amylase" (glucan 1,4-alpha-maltohydrolase, E.C.
3.2.1.133) is able to hydrolyze amylose and amylopectin to maltose
in the alpha-configuration. A maltogenic alpha-amylase from
Bacillus stearothermophilus strain NCIB 11837 is commercially
available from Novozymes A/S, Denmark. The maltogenic alpha-amylase
is described in U.S. Pat. Nos. 4,598,048, 4,604,355 and 6,162,628,
which are hereby incorporated by reference.
Bacterial Hybrid Alpha-Amylases
[0088] A hybrid alpha-amylase specifically contemplated comprises
445 C-terminal amino acid residues of the Bacillus licheniformis
alpha-amylase (shown as SEQ ID NO: 4 in WO 99/19467) and the 37
N-terminal amino acid residues of the alpha-amylase derived from
Bacillus amyloliquefaciens (shown as SEQ ID NO: 3 in WO 99/194676),
with one or more, especially all, of the following
substitution:
[0089] G48A+T49I+G107A+H156Y+A181T+N190F+I201F+A209V+Q264S (using
the Bacillus licheniformis numbering). Also preferred are variants
having one or more of the following mutations (or corresponding
mutations in other Bacillus alpha-amylase backbones): H154Y, A181T,
N190F, A209V and Q264S and/or deletion of two residues between
positions 176 and 179, preferably deletion of E178 and G179 (using
the SEQ ID NO: 5 numbering of WO 99/19467).
[0090] The bacterial alpha-amylase may be added in amounts as are
well-known in the art. When measured in KNU units (described below
in the "Materials & Methods"-section) the alpha-amylase
activity is preferably present in an amount of 0.5-5,000 NU/g of
DS, in an amount of 1-500 NU/g of DS, or more preferably in an
amount of 5-1,000 NU/g of DS, such as 10-100 NU/g DS.
Fungal Alpha-Amylases
[0091] Fungal acid alpha-amylases include acid alpha-amylases
derived from a strain of the genus Aspergillus, such as Aspergillus
oryzae, Aspergillus niger, Aspergillus kawachii alpha-amylases.
[0092] A preferred acid fungal alpha-amylase is a Fungamyl-like
alpha-amylase which is preferably derived from a strain of
Aspergillus oryzae. In the present disclosure, the term
"Fungamyl-like alpha-amylase" indicates an alpha-amylase which
exhibits a high identity, i.e. more than 70%, more than 75%, more
than 80%, more than 85% more than 90%, more than 95%, more than
96%, more than 97%, more than 98%, more than 99% or even 100%
identity to the mature part of the amino acid sequence shown in SEQ
ID NO: 10 in WO 96/23874.
[0093] Another preferred acid alpha-amylase is derived from a
strain Aspergillus niger. In a preferred embodiment the acid fungal
alpha-amylase is the one from Aspergillus niger disclosed as
"AMYA_ASPNG" in the Swiss-prot/TeEMBL database under the primary
accession no. P56271 and described in more detail in WO 89/01969
(Example 3). The acid Aspergillus niger acid alpha-amylase is also
shown as SEQ ID NO: 1 in WO 2004/080923 (Novozymes) which is hereby
incorporated by reference. Also variants of said acid fungal
amylase having at least 70% identity, such as at least 80% or even
at least 90% identity, such as at least 95%, at least 96%, at least
97%, at least 98%, or at least 99% identity to SEQ ID NO: 1 in WO
2004/080923 are contemplated. A suitable commercially available
acid fungal alpha-amylase derived from Aspergillus niger is SP288
(available from Novozymes A/S, Denmark).
[0094] In a preferred embodiment the alpha-amylase is derived from
Aspergillus kawachii and disclosed by Kaneko et al., 1996, J.
Ferment. Bioeng. 81:292-298, "Molecular-cloning and determination
of the nucleotide-sequence of a gene encoding an acid-stable
alpha-amylase from Aspergillus kawachii"; and further as
EMBL:#AB008370.
[0095] The fungal acid alpha-amylase may also be a wild-type enzyme
comprising a carbohydrate-binding module (CBM) and an alpha-amylase
catalytic domain (i.e., a none-hybrid), or a variant thereof. In an
embodiment the wild-type acid alpha-amylase is derived from a
strain of Aspergillus kawachii.
Fungal Hybrid Alpha-Amylases
[0096] In a preferred embodiment the fungal acid alpha-amylase is a
hybrid alpha-amylase. Preferred examples of fungal hybrid
alpha-amylases include the ones disclosed in WO 2005/003311 or U.S.
Patent Application Publication no. 2005/0054071 (Novozymes) or U.S.
patent application No. 60/638,614 (published as WO 2006/069290)
which is hereby incorporated by reference. A hybrid alpha-amylase
may comprise an alpha-amylase catalytic domain (CD) and a
carbohydrate-binding domain/module (CBM), such as a starch binding
domain, and optional a linker.
[0097] Specific examples of contemplated hybrid alpha-amylases
include those disclosed in Tables 1 to 5 of the examples in
co-pending U.S. patent application No. 60/638,614, including
Fungamyl variant with catalytic domain JA118 and Athelia rolfsii
SBD (SEQ ID NO: 100 in U.S. 60/638,614), Rhizomucor pusillus
alpha-amylase with Athelia rolfsii AMG linker and SBD (SEQ ID NO:
101 in U.S. 60/638,614), Rhizomucor pusillus alpha-amylase with
Aspergillus niger glucoamylase linker and SBD (which is disclosed
in Table 5 as a combination of amino acid sequences SEQ ID NO: 20,
SEQ ID NO: 72 and SEQ ID NO: 96 in U.S. application Ser. No.
11/316,535) or as V039 in Table 5 in WO 2006/069290, and Meripilus
giganteus alpha-amylase with Athelia rolfsii glucoamylase linker
and SBD (SEQ ID NO: 102 in U.S. 60/638,614). Other specifically
contemplated hybrid alpha-amylases are any of the ones listed in
Tables 3, 4, 5, and 6 in Example 4 in U.S. application Ser. no.
11/316,535 or WO 2006/069290 (hereby incorporated by
reference).
[0098] Other specific examples of contemplated hybrid
alpha-amylases include those disclosed in U.S. Patent Application
Publication no. 2005/0054071, including those disclosed in Table 3
on page 15, such as Aspergillus niger alpha-amylase with
Aspergillus kawachii linker and starch binding domain.
[0099] Contemplated are also alpha-amylases which exhibit a high
identity to any of above mention alpha-amylases, i.e., more than
70%, more than 75%, more than 80%, more than 85% more than 90%,
more than 95%, more than 96%, more than 97%, more than 98%, more
than 99% or even 100% identity to the mature enzyme sequences.
Commercial Alpha-Amylase Products
[0100] Preferred commercial compositions comprising alpha-amylase
include MYCOLASE from DSM (Gist Brocades), BAN.TM., TERMAMYL.TM.
SC, FUNGAMYL.TM., LIQUOZYME.TM. X and SAN.TM. SUPER, SAN.TM. EXTRA
L (Novozymes NS) and CLARASE.TM. L-40,000, DEX-LO.TM., SPEZYME.TM.
FRED, SPEZYME.TM. AA, and SPEZYME.TM. DELTA AA (Genencor Int.), and
the acid fungal alpha-amylase sold under the trade name SP288
(available from Novozymes A/S, Denmark).
[0101] An acid alpha-amylases may according to the invention be
added in an amount of 0.1 to 10 AFAU/g DS, preferably 0.10 to 5
AFAU/g DS, especially 0.3 to 2 AFAU/g DS.
Carbohydrate-Source Generating Enzymes
[0102] The term "carbohydrate-source generating enzyme" includes
glucoamylase (being glucose generators), beta-amylase and
maltogenic amylase (being maltose generators). A
carbohydrate-source generating enzyme is capable of producing a
carbohydrate that can be used as an energy-source by the fermenting
organism(s) in question, for instance, when used in a process of
the invention for producing a fermentation product, such as
ethanol. The generated carbohydrate may be converted directly or
indirectly to the desired fermentation product, preferably ethanol.
According to the invention a mixture of carbohydrate-source
generating enzymes may be used. Especially contemplated mixtures
are mixtures of at least a glucoamylase and an alpha-amylase,
especially an acid amylase, even more preferred an acid fungal
alpha-amylase. The ratio between acidic fungal alpha-amylase
activity (AFAU) per glucoamylase activity (AGU) (AFAU per AGU) may
in an embodiment of the invention be at least 0.1, in particular at
least 0.16, such as in the range from 0.12 to 0.50 or more.
Glucoamylases
[0103] A glucoamylase used according to the invention may be
derived from any suitable source, e.g., derived from a
micro-organism or a plant. Preferred glucoamylases are of fungal or
bacterial origin, e.g., selected from the group consisting of
Aspergillus glucoamylases, in particular A. niger G1 or G2
glucoamylase (Boel et al., 1984, EMBO J. 3(5): 1097-1102), or
variants thereof, such as one disclosed in WO 92/00381, WO
00/04136, WO 01/04273 and WO 03/029449 (from Novozymes, Denmark,
hereby incorporated by reference); the A. awamori glucoamylase (WO
84/02921), A. oryzae (Agric. Biol. Chem. 55(4): 941-949 (1991)), or
variants or fragments thereof.
[0104] Other Aspergillus glucoamylase variants include variants to
enhance the thermal stability: G137A and G139A (Chen et al., 1996,
Prot. Eng. 9: 499-505); D257E and D293E/Q (Chen et al., 1995, Prot.
Engng. 8: 575-582); N182 (Chen et al., 1994, Biochem. J. 301:
275-281); disulphide bonds, A246C (Fierobe et al., 1996,
Biochemistry 35: 8698-8704; and introduction of Pro residues in
position A435 and S436 (Li et al., 1997, Protein Engng. 10:
1199-1204. Other glucoamylases include Athelia rolfsii (previously
denoted Corticium rolfsii) glucoamylase (see U.S. Pat. No.
4,727,026 and (Nagasaka et al., 1998, Purification and properties
of the raw-starch-degrading glucoamylases from Corticium rolfsii,
Appl. Microbiol. Biotechnol. 50:323-330), Talaromyces
glucoamylases, in particular, derived from Talaromyces emersonii
(WO 99/28448), Talaromyces leycettanus (U.S. Pat. No. Re. 32,153),
Talaromyces duponti, Talaromyces thermophilus (U.S. Pat. No.
4,587,215), or Trametes cingulata (WO 2006/069289). Bacterial
glucoamylases contemplated include glucoamylases from the genus
Clostridium, in particular C. thermoamylolyticum (EP 135,138), and
C. thermohydrosulfuricum (WO 86/01831).
[0105] Commercially available compositions comprising glucoamylase
include AMG 200L; AMG 300 L; SAN.TM. SUPER, SAN.TM. EXTRA L,
SPIRIZYME.TM. PLUS, SPIRIZYME.TM. FUEL, SPIRIZYME.TM. B4U and
AMG.TM. E (from Novozymes NS); OPTIDEX.TM. 300 (from Genencor
Int.); AMIGASE.TM. and AMIGASE.TM. PLUS (from DSM); G-ZYME.TM.
G900, G-ZYME.TM. and G990 ZR (from Genencor Int.).
[0106] Glucoamylase may in an embodiment be added in an amount of
0.005-5 AGU/g DS, more preferably between 0.01-1 AGU/g DS, such as
especially around 0.1-0.5 AGU/g DS.
Beta-Amylases
[0107] At least according to the invention the a beta-amylase (E.0
3.2.1.2) is the name traditionally given to exo-acting maltogenic
amylases, which catalyze the hydrolysis of 1,4-alpha-glucosidic
linkages in amylose, amylopectin and related glucose polymers.
Maltose units are successively removed from the non-reducing chain
ends in a step-wise manner until the molecule is degraded or, in
the case of amylopectin, until a branch point is reached. The
maltose released has the beta anomeric configuration, hence the
name beta-amylase.
[0108] Beta-amylases have been isolated from various plants and
microorganisms (W. M. Fogarty and C. T. Kelly, Progress in
Industrial Microbiology, vol. 15, pp. 112-115, 1979). These
beta-amylases are characterized by having optimum temperatures in
the range from 40.degree. C. to 65.degree. C. and optimum pH in the
range from 4.5 to 7. A commercially available beta-amylase from
barley is NOVOZYM.TM. WBA from Novozymes NS, Denmark and
SPEZYME.TM. BBA 1500 from Genencor Int., USA.
Maltogenic Amylases
[0109] The amylase may also be a maltogenic alpha-amylase. A
"maltogenic alpha-amylase" (glucan 1,4-alpha-maltohydrolase, E.C.
3.2.1.133) is able to hydrolyze amylose and amylopectin to maltose
in the alpha-configuration. A maltogenic amylase from Bacillus
stearothermophilus strain NCIB 11837 is commercially available from
Novozymes NS. Maltogenic alpha-amylases are described in U.S. Pat.
Nos. 4,598,048, 4,604,355 and 6,162,628, which are hereby
incorporated by reference.
[0110] The maltogenic amylase may in a preferred embodiment be
added in an amount of 0.05-5 mg total protein/gram DS or 0.05-5
MANU/g DS.
Use of Antibacterial Agents
[0111] In the final aspect the invention relates to the use of
antibacterial agents for killing and/or inhibiting bacterial growth
in fermentation product production processes.
[0112] The antibacterial agent is any of the ones mentioned in the
"Antibacterial Agents" section above. Preferred are peptides,
polypeptides, proteins and enzyme, preferably of bacterial or
fungal origin or prepared synthetically.
Materials and Methods
Enzymes:
[0113] Bacterial Alpha-amylase A: Bacillus stearothermophilus
alpha-amylase variant with the mutations: I181*+G182*+N193F
disclosed in U.S. Pat. No. 6,187,576 and available on request from
Novozymes NS, Denmark. [0114] Glucoamylase T: Glucoamylase derived
from Talaromyces emersonii disclosed in WO1999/028448 and available
from Novozymes NS, Denmark.
Antibacterial Agents:
[0114] [0115] Peptide A: Fungal defensin derived from the
saprophytic ascomycete Pseudoplectania nigrella also disclosed as
amino acids 1-40 in SEQ ID NO: 2 in WO 03/044049. [0116] Peptide B:
Synthetic peptide disclosed as SEQ ID NO:93 in WO 2005/105831.
[0117] Lysozyme from hen egg white, cat #L-7651, lot #114K7054
purchased from Sigma.
Yeast:
[0117] [0118] RED STAR.RTM. available from Red Star/Lesaffre,
USA
Bacteria:
[0119] Three of the Lactobacillus strains used in this study (L.
plantarum #1, L. paracasei #2, L. paracasei #2a) were kindly
donated by Professor Mike Ingledew (U. of Saskatchewan).
Media
[0120] CASO: Tryptic soy broth from BD Bacto Ref 211822 [0121] MRS
agar (EMD Science, 1.10660.0500)
Equipment:
[0121] [0122] Spectrophotometer: Tecan Safire Austria, Serial No.
12901300079
Methods:
Lactobacillus Samples
[0123] The Lactobacillus strain (e.g., L. plantarum #1, L.
paracasei #2, L. paracasei #2a) is stored as frozen culture until
use. See J. Appl. Microbiol. 90: 819-28 (2001) for details. The
culture is rehydrated in MRS broth (Difco) to initial cell
concentrations of around 4.times.10.sup.7. Samples are plated on
MRS agar (EMD Science, 1.10660.0500) in an anaerobic environment
for 2 days at 37.degree. C. for colony counting.
Alpha-Amylase Activity (KNU)
[0124] The amylolytic activity may be determined using potato
starch as substrate. This method is based on the break-down of
modified potato starch by the enzyme, and the reaction is followed
by mixing samples of the starch/enzyme solution with an iodine
solution. Initially, a blackish-blue color is formed, but during
the break-down of the starch the blue color gets weaker and
gradually turns into a reddish-brown, which is compared to a
colored glass standard.
[0125] One Kilo Novo alpha amylase Unit (KNU) is defined as the
amount of enzyme which, under standard conditions (i.e. at
37.degree. C.+/-0.05; 0.0003 M Ca.sup.2+; and pH 5.6) dextrinizes
5260 mg starch dry substance Merck Amylum solubile.
[0126] A folder EB-SM-0009.02/01 describing this analytical method
in more detail is available upon request to Novozymes A/S, Denmark,
which folder is hereby included by reference.
Determination of Acid Alpha-Amylase Activity (AFAU)
[0127] Acid alpha-amylase activity is measured in AFAU (Acid Fungal
Alpha-amylase Units), which are determined relative to an enzyme
standard.
[0128] The standard used is AMG 300 L (from Novozymes NS, Denmark,
glucoamylase wild-type Aspergillus niger G1, also disclosed in Boel
et al., 1984, EMBO J. 3 (5): 1097-1102) and WO 92/00381). The
neutral alpha-amylase in this AMG falls after storage at room
temperature for 3 weeks from approx. 1 FAU/mL to below 0.05
FAU/mL.
[0129] The acid alpha-amylase activity in this AMG standard is
determined in accordance with the following description. In this
method, 1 AFAU is defined as the amount of enzyme, which degrades
5.260 mg starch dry matter per hour under standard conditions.
[0130] Iodine forms a blue complex with starch but not with its
degradation products. The intensity of color is therefore directly
proportional to the concentration of starch. Amylase activity is
determined using reverse colorimetry as a reduction in the
concentration of starch under specified analytic conditions.
##STR00001##
[0131] Standard conditions/reaction conditions: (per minute)
[0132] Substrate: Starch, approx. 0.17 g/L
[0133] Buffer: Citate, approx. 0.03 M
[0134] Iodine (I.sub.2): 0.03 g/L
[0135] CaCl.sub.2: 1.85 mM
[0136] pH: 2.50.+-.0.05
[0137] Incubation temperature: 40.degree. C.
[0138] Reaction time: 23 seconds
[0139] Wavelength: lambda=590 nm
[0140] Enzyme concentration: 0.025 AFAU/mL
[0141] Enzyme working range: 0.01-0.04 AFAU/mL
[0142] If further details are preferred these can be found in
EB-SM-0259.02/01 available on request from Novozymes NS, Denmark,
and incorporated by reference.
Acid Alpha-Amylase Units (AAU)
[0143] The acid alpha-amylase activity can be measured in AAU (Acid
Alpha-amylase Units), which is an absolute method. One Acid Amylase
Unit (AAU) is the quantity of enzyme converting 1 g of starch (100%
of dry matter) per hour under standardized conditions into a
product having a transmission at 620 nm after reaction with an
iodine solution of known strength equal to the one of a color
reference.
[0144] Standard conditions/reaction conditions:
[0145] Substrate: Soluble starch. Concentration approx. 20 g
DS/L.
[0146] Buffer: Citrate, approx. 0.13 M, pH=4.2
[0147] Iodine solution: 40.176 g potassium iodide+0.088 g
iodine/L
[0148] City water 15.degree.-20.degree. dH (German degree
hardness)
[0149] pH: 4.2
[0150] Incubation temperature: 30.degree. C.
[0151] Reaction time: 11 minutes
[0152] Wavelength: 620 nm
[0153] Enzyme concentration: 0.13-0.19 AAU/mL
[0154] Enzyme working range: 0.13-0.19 AAU/mL
[0155] The starch should be Lintner starch, which is a thin-boiling
starch used in the laboratory as colorimetric indicator. Lintner
starch is obtained by dilute hydrochloric acid treatment of native
starch so that it retains the ability to color blue with iodine.
Further details can be found in EP 0140410B2, which disclosure is
hereby included by reference.
Glucoamylase Activity (AGI)
[0156] Glucoamylase (equivalent to amyloglucosidase) converts
starch into glucose. The amount of glucose is determined here by
the glucose oxidase method for the activity determination. The
method described in the section 76-11 Starch--Glucoamylase Method
with Subsequent Measurement of Glucose with Glucose Oxidase in
"Approved methods of the American Association of Cereal Chemists".
Vol. 1-2 AACC, from American Association of Cereal Chemists, 2000;
ISBN: 1-891127-12-8.
[0157] One glucoamylase unit (AGI) is the quantity of enzyme which
will form 1 micromol of glucose per minute under the standard
conditions of the method.
[0158] Standard conditions/reaction conditions: [0159] Substrate:
Soluble starch. [0160] Concentration approx. 16 g dry matter/L.
[0161] Buffer: Acetate, approx. 0.04 M, pH=4.3 [0162] pH: 4.3
[0163] Incubation temperature: 60.degree. C. [0164] Reaction time:
15 minutes [0165] Termination of the reaction: NaOH to a
concentration of approximately 0.2 g/L (pH-9) [0166] Enzyme
concentration: 0.15-0.55 AAU/mL.
[0167] The starch should be Lintner starch, which is a thin-boiling
starch used in the laboratory as colorimetric indicator. Lintner
starch is obtained by dilute hydrochloric acid treatment of native
starch so that it retains the ability to color blue with
iodine.
Glucoamylase Activity (AGU)
[0168] The Novo Glucoamylase Unit (AGU) is defined as the amount of
enzyme, which hydrolyzes 1 micromole maltose per minute under the
standard conditions 37.degree. C., pH 4.3, substrate: maltose 23.2
mM, buffer: acetate 0.1 M, reaction time 5 minutes.
[0169] An autoanalyzer system may be used. Mutarotase is added to
the glucose dehydrogenase reagent so that any alpha-D-glucose
present is turned into beta-D-glucose. Glucose dehydrogenase reacts
specifically with beta-D-glucose in the reaction mentioned above,
forming NADH which is determined using a photometer at 340 nm as a
measure of the original glucose concentration.
TABLE-US-00001 AMG incubation: Substrate: maltose 23.2 mM Buffer:
acetate 0.1M pH: 4.30 .+-. 0.05 Incubation temperature: 37.degree.
C. .+-. 1 Reaction time: 5 minutes Enzyme working range: 0.5-4.0
AGU/mL Color reaction: GlucDH: 430 U/L Mutarotase: 9 U/L NAD: 0.21
mM Buffer: phosphate 0.12M; 0.15M NaCl pH: 7.60 .+-. 0.05
Incubation temperature: 37.degree. C. .+-. 1 Reaction time: 5
minutes Wavelength: 340 nm
[0170] A folder (EB-SM-0131.02/01) describing this analytical
method in more detail is available on request from Novozymes NS,
Denmark, which folder is hereby included by reference.
Determination of Maltoqenic Amylase Activity (MANU)
[0171] One MANU (Maltogenic Amylase Novo Unit) may be defined as
the amount of enzyme required to release one micro mole of maltose
per minute at a concentration of 10 mg of maltotriose (Sigma M
8378) substrate per ml of 0.1 M citrate buffer, pH 5.0 at
37.degree. C. for 30 minutes.
EXAMPLES
Example 1
MIC Test for Two Antibacterial Peptides on Four Lactobacillus
Cultures
[0172] Two antibacterial peptides (Peptide A and Peptide B) were
tested using MIC test (Minimal Inhibitory Concentration).
[0173] Four different Lactobacillus cultures were prepared in CASO
medium for 2 days at 37.degree. C. under facultative anaerobic
conditions. The four Lactobacillus cultures (Lactobacillus
paracasei #2a, Lactobacillus paracasei #2, Lactobacillus plantarum
#1, and Lactobacillus fermentum ATCC 14931) were mixed in equal
volume. 190 microL bacterial culture was filled into microtiter
plate wells (96 plate wells) and 10 microL of Peptide A and Peptide
B, respectively, were added (total well volume was then 200
microL). Measurements were initiated. The microtiter plates were
incubated with lids for 24 hours at 37.degree. C. to secure optimal
growth of facultative anaerobic lactobacilli strains. Peptide A and
Peptide B were prepared from initial protein concentration of 4.27
mg protein/mL and 34.34 mg protein/mL respectively. The tested
dosages were 0.5 to 60 micro grams peptide/ml for both peptides.
All treatments were run in 8 replicates and the control
(cells+media) were tested in 16 replicates.
[0174] FIGS. 1 and 2 display the result of MIC test determined at
600 nm using a spectrophotometer.
Example 2
Antibacterial Sffect of Peptide A in SSF.
[0175] Milled corn was liquefied in an aqueous slurry (pH 5.6)
using 50 NU/g DS Bacterial Alpha-Amylase A by heating until a
temperature of 85.degree. C. (approx 20 minutes) was reached.
Thereafter the slurry is cooked for another 60 minutes.
[0176] The corn mash (CM) was split up into approximately 5 g of CM
and added to 15 mL plastic centrifuge tubes. Fermentations were
carried out as SSF at 32.degree. C., 70 hours using RED STAR.RTM.
yeast at a dosage around 1.times.10.sup.7 Cells/ml of mash. Prior
to the start of the fermentations, a 1/1/1 mixture of three
Lactobacillus strains (Lactobacillus paracasei #2, Lactobacillus
paracasei #2a, Lactobacillus plantarum) were added to some of the
tubes containing the corn mash as indicated in Table 1 below and
allowed to grow for 24 hours at 32.degree. C. prior to pitching the
yeast. The target initial total Lactobacillus cell count was
1.times.10.sup.7 Cells/ml of mash, containing roughly equal cell
counts of each strain. All tests were each run in 9 replicates and
controls were included in the fermentation. The dry solid load was:
32.68 wt. %. The fermentations were monitored by weighing the
individual tubes and recording the time and date of the
measurement. The fermentation data was transferred to SAS JMP for
conducting analysis of variance, test carried out using
(.alpha.=0.05).
TABLE-US-00002 Glucoamylase T Peptide A Treatment AGU/g DS
microgram/g DS Lactobacillus Control 0.500 PL 1 0.500 1 + PL 5
0.500 5 + PL 25 0.500 25 + Control_Lb 0.500 +
[0177] Lactobacillus strains were cultured in standard CASO
broth/TSB, and incubated for two days in facultative anaerobic
conditions at 37.degree. C. Lactobacillus MRS Agar (EMD Science,
1.10660.0500) was used for plate counting. After plating 1 ml of
the dilutions mentioned in Table 2 and displayed in FIG. 3, the
plates were incubated in anaerobic conditions for 2 days at
37.degree. C.
[0178] The yeast counts were done by plating 1 ml of the dilutions
mentioned in Table 5 on Yeast and Mold Petrifilm Plates (3M,6407)
and incubated for 2 days at 32.5.degree. C.
TABLE-US-00003 TABLE 2 List of Lactobacillus counts over 3 days,
Final counts (cfu/mL). 0 hours 24 hours 48 hours 72 hours Control
10000 9.1E+06 0.0E+00 1.2E+08 PL1 1.8E+06 1.7E+09 1.8E+08 1.8E+06
PL5 9.1E+05 1.9E+09 1.3E+08 6.4E+04 PL25 9.1E+05 1.5E+09 1.3E+08
9.1E+05 Control Lb 1.4E+07 2.1E+09 1.7E+08 6.0E+06
Example 3
Effect of Lysozyme on Lactobacillus in Fermentation Medium
[0179] Milled corn was liquefied in an aqueous slurry (pH 5.6)
using 50 NU/g DS Bacterial Alpha-Amylase A by heating until a
temperature of 85.degree. C. (approx 20 minutes) was reached.
Thereafter the slurry is cooked for another 60 minutes. A sample of
the liquefied corn mash (CM) was pH-adjusted to 5.05 with
H.sub.2SO.sub.4. After pH adjustment, the mash was split up into
approximately 5 g of CM and added to 36 15 mL plastic centrifuge
tubes. Various amounts of L. paracasei were then added to give an
initial cell count of about 1-3.times.10.sup.6/mL. Different
dosages (0-100-300-1000 mg/L) of Lysozyme (Sigma) were also added
to each tube. The tubes were then thoroughly vortexed and placed in
a rack in 32.degree. C. water bath. Tubes were pulled after 0, 24,
and 48 hours for bacterial plating and counting. No additional
Lysozyme was added to any of the tubes after time zero.
[0180] FIG. 4 display the result of the test. The data suggests
that Lysozyme is effective inhibiting growth of Lactibacillus
paracasei.
* * * * *