U.S. patent application number 14/627753 was filed with the patent office on 2015-09-17 for fermentation processes and by-products.
This patent application is currently assigned to DIREVO INDUSTRIAL BIOTECHNOLOGY GMBH. The applicant listed for this patent is Leonie Degener, Christian Elend, Steffen Koehler, Klaudija Milos. Invention is credited to Leonie Degener, Christian Elend, Steffen Koehler, Klaudija Milos.
Application Number | 20150259633 14/627753 |
Document ID | / |
Family ID | 45558667 |
Filed Date | 2015-09-17 |
United States Patent
Application |
20150259633 |
Kind Code |
A1 |
Milos; Klaudija ; et
al. |
September 17, 2015 |
FERMENTATION PROCESSES AND BY-PRODUCTS
Abstract
Methods of improving the quality of by-products or residues
derived from starch-containing material in a processes for
producing fermentation products including adding an enzyme
composition comprising an enzyme or a mixture of enzymes capable of
degrading one or more fermented mash components to fermented mash;
and separating fermentation product.
Inventors: |
Milos; Klaudija; (Cologne,
DE) ; Koehler; Steffen; (Cologne, DE) ; Elend;
Christian; (Cologne, DE) ; Degener; Leonie;
(Cologne, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Milos; Klaudija
Koehler; Steffen
Elend; Christian
Degener; Leonie |
Cologne
Cologne
Cologne
Cologne |
|
DE
DE
DE
DE |
|
|
Assignee: |
DIREVO INDUSTRIAL BIOTECHNOLOGY
GMBH
Cologne
DE
|
Family ID: |
45558667 |
Appl. No.: |
14/627753 |
Filed: |
February 20, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13995079 |
Aug 19, 2013 |
8962286 |
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PCT/EP2011/006473 |
Dec 21, 2011 |
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14627753 |
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61425893 |
Dec 22, 2010 |
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Current U.S.
Class: |
426/13 ; 426/28;
426/52; 426/53; 435/200; 435/209 |
Current CPC
Class: |
A23K 50/30 20160501;
Y02E 50/10 20130101; C12Y 302/01004 20130101; C12Y 302/01015
20130101; C12N 9/14 20130101; Y02P 60/87 20151101; C12N 9/2402
20130101; Y02E 50/17 20130101; Y02P 60/873 20151101; A23K 50/75
20160501; C12N 9/2437 20130101; A23K 10/38 20160501; C12F 3/10
20130101; C12N 9/244 20130101; C12P 7/14 20130101; C12P 7/06
20130101; C12N 9/2491 20130101; C12C 7/053 20130101; C12N 9/2482
20130101 |
International
Class: |
C12F 3/10 20060101
C12F003/10; C12N 9/42 20060101 C12N009/42; C12N 9/24 20060101
C12N009/24; A23K 1/06 20060101 A23K001/06; C12C 7/053 20060101
C12C007/053 |
Claims
1. A method to improve the quality of by-products or residues
derived from starch-containing material in a processes for
producing fermentation products comprising the steps of: a) adding
an enzyme composition comprising an enzyme or a mixture of enzymes
capable of degrading one or more fermented mash components to
fermented mash; and b) separating fermentation product from
degraded mash.
2. The method according to claim 1, wherein the enzyme composition
comprises an enzyme selected from the group consisting of an
amylase, alpha-amylase, glucoamylase, a cellulase, a
beta-glucanase, a hemicellulase, a xylanase, a pectinase, a
mannanase, and a protease, and a mixture thereof.
3. The method according to claim 1, wherein the enzyme composition
comprises a member selected from the group consisting of: a) a
beta-1,3-glucanase; b) a beta-1,3-glucanase and a
1,6-beta-glucanase; c) a xylanase and optionally a
beta-1,3-glucanase; d) a beta-1,3-glucanase, a 1,6-beta-glucanase,
and a xylanase, e) a beta-1,3-glucanase, a xylanase and a
pectinase; f) a beta-1,3-glucanase, a xylanase and a protease; g) a
mannanase; and h) a mannanase and a beta-1,3-glucanase.
4. The method according to claim 1, wherein the fermentation
product is selected from the group consisting of an acid, an
alcohol and hydrogen.
5. The method according to claim 4, wherein the alcohol is selected
from the group consisting of ethanol, butanol, propanol, methanol,
propanediol and butanediol.
6. The method according to claim 1, wherein the fermented mash is
derived from a process of producing a fermentation product
utilizing sugar-containing material as feedstock.
7. The method according to claim 1, wherein the fermented mash is
derived from a process of producing a fermentation product
utilizing starch-containing material as feedstock.
8. The method according to claim 1, wherein the fermented mash is
derived from a process of producing a fermentation product
utilizing a cereal feedstock.
9. The method according to claim 1, wherein the by-product or
residue is a fibrous by-product selected from the group consisting
of spent brewer's grains, dried distiller's grains, dried
distiller's soluble, distiller's dried grains with soluble, wet
grains, and mixtures thereof.
10. The method according to claim 1, wherein the fermented mash is
derived from a process of producing a fermentation product
utilizing whole grain obtained from cereals as feedstock, the
fermentation product is ethanol, the ethanol is separated by
distillation and the improved by-product is Dried Distillers Grains
with Solubles (DDGS).
11. The method according to claim 1, wherein by-products or
residues have an improved nutrition quality and are used in animal
feed.
12. A method of producing ethanol from starch containing material,
the method comprising the steps of: a) converting starch containing
material to fermentable sugars; b) fermenting fermentable sugars
with a microorganism to fermented mash; c) subjecting the fermented
mash after fermentation to an enzyme composition comprising an
enzyme or a mixture of enzymes; d) separating ethanol from the
fermented mash by distillation
13. The method according to claim 12, wherein the enzyme
composition comprises an enzyme selected from the group consisting
of amylase, an alpha-amylase, glucoamylase, cellulase,
beta-glucanase, hemicellulase, xylanase, pectinase, mannanase, a
protease, and a mixture thereof.
14. The method according to claim 12, wherein the enzyme
composition comprises a member selected from the group consisting
of a) beta-1,3-glucanase; b) xylanase; c) beta-1,3-glucanase and a
xylanase; d) beta-1,3-glucanase, xylanase and a pectinase; e)
beta-1,3-glucanase, a xylanase and a protease; f) a mannanase; g)
mannanase and a beta-1,3-glucanase.
15. The method according to claim 12, wherein the starch containing
material is obtained from cereals and/or tubers.
16. The method according to claim 12, wherein the by-product or
residue is a fibrous by-product selected from the group consisting
of spent brewer's grains, dried distiller's grains, dried
distiller's soluble, distiller's dried grains with soluble, wet
grains, and mixtures thereof.
17. A method for manufacturing an enzyme composition used for
treating fermented mash of in a fermentation production process to
improve the nutritional quality of a by-product or residue and/or
the processability of the production process, comprising: a)
inoculating the by-product or residue with at least one filamentous
fungus; b) fermenting the by-product or residue; and c) separating
at least one enzyme from the fermented by-product or residue.
18. The method according to claim 17, wherein the filamentous
fungus is selected from the group consisting of Rhizopus,
Aspergillus, Trichoderma, and a combination thereof.
19. The method according to claim 17, wherein the by-product or
residue is a fibrous by-product selected from the group consisting
of spent brewer's grains, dried distillier's grains, dried
distiller's soluble, distillers dried grains with soluble, wet
grains, and mixtures thereof.
Description
FIELD OF THE INVENTION
[0001] The present disclosure relates to an improved process of
producing a fermentation product, in particular ethanol. The
present disclosure relates also to the use of enzymes for improving
the quality of by-products in the fermentative production process
and to compositions comprising enzymes capable of degrading
components in the fermented mash in the fermentation process.
BACKGROUND OF THE INVENTION
[0002] Fermentation products, such as ethanol, are produced by
first degrading starch-containing material into fermentable sugars
by liquefaction and saccharification and then converting the sugars
directly or indirectly into the desired fermentation product using
a fermenting organism. Liquid fermentation products such as ethanol
are recovered from the fermented mash (often referred to as "beer"
or "beer mash"), e.g., by distillation, which separate the desired
fermentation product from other liquids and/or solids. The
remaining faction, referred to as "whole stillage", is dewatered
and separated into a solid and a liquid phase, e.g., by
centrifugation. The solid phase is referred to as "wet cake" (or
"wet grains" or "WDG") and the liquid phase (supernatant) is
referred to as "thin stillage". Dewatered wet cake is dried to
provide "Distillers Dried Grains" (DDG) used as nutrient in animal
feed. Thin stillage is typically evaporated to provide condensate
and syrup (or "thick stillage") or may alternatively be recycled
directly to the slurry tank as "backset". Condensate may either be
forwarded to a methanator before being discharged or may be
recycled to the slurry tank. The syrup consisting mainly of limit
dextrins and non-fermentable sugars may be blended into DDG or
added to the wet cake before drying to produce DDGS (Distillers
Dried Grain with Solubles).
[0003] It is known to commercially use the various byproducts and
residues derived from the fermentation processes like the ethanol
production process. Distillers residues or byproducts, as well as
by-products of cereal and other food industry manufacturing, are
known to have a certain value as sources of protein and energy for
animal feed. Furthermore, the oil from the by-products like DDGS
can be recovered as a separate by-product for use in biodiesel
production or other biorenewable products are sought.
[0004] The by-products like DDG, DDGS or WDG comprises proteins,
fibers, fat and unconverted starch. For example DDGS contains
typically about 30% of protein. While the protein content is high
the amino acid composition is not well suited for monogastric
animals if used as animal feed. In general processing of DDGS,
especially drying time and temperature are effecting the
availability and digestibility of the amino acids, especially
lysine.
[0005] Furthermore, the by-products are mainly fibrous by-products
comprising Crude Fibers (CF), which are structural carbohydrates
consisting of cellulose, hemicellulose and indigestible materials
like lignin. The structural carbohydrates are not digestible in
animal's small intestine. Fibers are characterized and analyzed by
different methods and can be divided into crude fibers (CF),
neutral detergent fibers (NDF) and acid detergent fibers (ADF). The
proportion of cellulose and lignin in the crude fibers fraction
also determines the digestibility of crude fibers or its solubility
in the intestine. High cellulose and lignin concentrations mean
reduced digestibility and vice versa. Hemicelluloses are capable to
bind water. The soluble part of fibres the soluble
non-starch-polysaccharides (NSP) cannot be digested by monogastric
animals like swine and poultry, but increase viscosity, due to
their capability to bind water, and are a nutritional constraint,
since they can cause moist, sticky droppings and wet litter. The
antinutritional effect of soluble NSP's is mainly related to the
increase in digesta viscosity. The increased viscosity is slowing
down the feed passage rate and hinders the intestinal uptake of
nutrients and can lead to decreased feed uptake The viscosity
increase a) hinders the intestinal absorption of nutrients and can
result in negative effect on the consistency on faces and even
symptoms of diarrhea, b) slowing down the feed passage rate and
possibly to decreased feed intake. Another effect of NSP's is the
so-called "Nutrient Encapsulation". The NSP's in plant cell wall
encapsulated starch, protein, oil and other nutrients within the
plant cell which is an impermeable barrier preventing full
utilization of the nutrients within the cell.
[0006] Furthermore the soluble NSP's are responsible for high
viscosities during fermentation and are directly influencing
separation and drying conditions of fermentation by-products like
DDGS in the production process. The bound or encapsulated water in
the product is difficult to remove and causes the use of higher
drying temperatures and also longer drying time, adversely
affecting the quality of temperature-sensitive products like amino
acids. The availability and digestibility of essential amino acids
in the by-products are lowered by high temperatures and long drying
time during production. Examples for NSPs are arabinoxylans,
beta-glucans, galactomannans and alpha-galactosides.
[0007] As the by-products are used in animal feed for monogastrics
animals like pigs and poultry it is important that the by-products
have high concentrations of protein with a good amino acid
composition and high availability and low soluble fibers
content.
[0008] Therefore, the two ways for an improvement of a fermentative
production plant ton increase their efficiency and profitability
are an improved production process and the improvement of the
quality of the by-products.
[0009] In the prior art, a lot of specific processes or treatment
methods are described to improve fermentative production
processes.
[0010] For example, WO 2007/056321 A1 discloses a method of
dewatering whole stillage comprising adding enzymes to whole
stillage in the ethanol production to improve the solid-liquid
separation in the process.
[0011] WO 02/38786 describes a process of ethanol production,
whereby enzymes are used for thinning the liquefied whole grain
mash and the thin stillage. Enzymes are applied to the liquefied
mash before the fermentation starts as well as to the thin stillage
after centrifugation of the whole stillage.
[0012] The US 2006/0275882 A1 describes a process for producing a
fermentation product wherein the viscosity of the mash is reduced
by the application of enzymes before or during the
fermentation.
[0013] The US 2006/0233864 A1 describes a method for improving the
nutritional quality of fibrous by-products for a food manufacturing
process, wherein the fibrous by-products like DDGS are inoculated
with a filamentous fungus to improve the quality of the
by-product.
[0014] Some ethanol plants use milo, wheat, or barley in the
fermentation process, depending on geographical location and time
of the year. As a result, nutrient composition can vary among DDGS
sources. Because of the near complete fermentation of starch, the
remaining amino acids, fat, minerals and vitamins increase
approximately three-fold in concentration compared to levels found
in corn. Despite the significant increase in crude protein, the
poor amino acid balance of DDGS must be addressed when formulating
swine and poultry diets.
[0015] Therefore, it is an object of the present disclosure to
provide improved methods for improving the quality of the
by-products from fermentation processes. It is further a need for
methods for further improvement of the process ability by
dewatering the stillage and to provide improved methods for
increasing the amount of recoverable oil.
SUMMARY OF THE DISCLOSURE
[0016] The present disclosure relates to methods for the
improvement of the quality of the by-products or residues derived
from fermented mash comprising the steps of: i) subjecting the
fermented mash during or after the fermentation to an enzyme
composition comprising an enzyme or a mixture of enzymes capable of
degrading one or more fermented mash components, ii) separating the
desired fermentation product.
[0017] The present disclosure also relates to methods of producing
ethanol from starch containing material, said method comprising the
steps of: [0018] i) Converting starch containing material to
fermentable sugars [0019] ii) Fermentation of the fermentable
sugars with a microorganism to fermented mash [0020] iii)
Subjecting the fermented mash after the fermentation process to an
enzyme composition comprising an enzyme or a mixture of enzymes
[0021] iv) Separation of the ethanol in the fermented mash by
distillation
[0022] The present disclosure also relates to uses of an enzyme
composition comprising a beta-1,3-glucanase and/or a xylanase for
the improvement of the nutritional quality of the by-products or
residues derived from fermented mash in a fermentative production
process.
[0023] The present disclosure relates to a process of fermenting a
starch-containing material into a fermentation product comprising a
fermentation step without the presence of a beta 1,3-glucanase
and/or a xylanase. After the fermentation an enzyme composition is
added to the fermented mash for an improvement of the by-products
like the fibrous by-products such as spent brewer's grains, dried
distiller's grains, dried distiller's soluble, distiller's dried
grains with soluble, wet grains, and mixtures thereof.
BRIEF DESCRIPTION OF THE DRAWING
[0024] FIG. 1 schematically shows an ethanol production
process.
[0025] FIG. 2 schematically shows an ethanol process including on
site fermentation tank for enzyme production based on WDG.
[0026] FIG. 3 schematically shows an ethanol process including on
site fermentation tank for enzyme production based on whole
stillage.
[0027] FIG. 4 is a diagram showing the reduction of ADF and NDF in
DDGS by using 1,3-.beta.-glucanase.
[0028] FIG. 5 is a diagram showing the reduction of ADF and NDF in
DDGS by using xylanase.
[0029] FIG. 6 is a diagram showing the reduction of ADF and NDF in
DDGS by using an enzyme composition comprising
1,3,-.beta.-glucanase and xylanase.
[0030] FIG. 7 is a diagram showing the reduction of ADF and NDF in
DDGS by using an enzyme composition comprising
1,3,-.beta.-glucanase and xylanase and a pectinase or a
protease.
[0031] FIG. 8 showing a picture of thin stillage from not enzyme
treated beer, whereby no oil separation can be shown.
[0032] FIG. 9 showing a picture of thin stillage from enzyme
treated beer, whereby a clear separation of the oil forming in
thick oil layer is shown.
[0033] FIG. 10 is a diagram showing the weight gain of quails fed
with different feed stuff.
[0034] FIG. 11 is a diagram showing the dewatering capabilities of
an enzyme based process according to the present disclosure by
using an enzyme composition comprising 1,3,-.beta.-glucanase and
xylanase.
[0035] FIG. 12 is a diagram showing an in vitro-digestibility assay
of DDGS produced using an enzyme composition comprising
1,3,-.beta.-glucanase and xylanase in a process according to the
present disclosure.
[0036] FIG. 13 is a diagram showing the improved protein
availability of DDGS produced using an enzyme composition
comprising 1,3,-.beta.-glucanase and xylanase in a process
according to the present disclosure.
DESCRIPTION OF THE INVENTION
[0037] The object of the present invention is to provide improved
fermentative production processes due to a better process ability
and to provide by-products from the fermentation process with an
improved quality.
[0038] One aspect of the present disclosure relates to methods for
improving the quality of by-products or residues derived from
starch-containing material in a processes for producing
fermentation products comprising the steps of: i) subjecting the
fermented mash after the fermentation to an enzyme composition
comprising an enzyme or a mixture of enzymes capable of degrading
one or more fermented mash components, ii) separating the desired
fermentation product.
[0039] By-products or residues of the fermenting process includes
distillers' grain, brewer's grains, dried distiller's grains, dried
distiller's solubles, distiller's dried grains with solubles, WDG
or/and residues of the cereal processing industry, or mixtures
thereof. For example, DDGS are the dried residue remaining after
the starch fraction of corn is fermented with selected yeasts and
enzymes to produce ethanol and carbon dioxide. After complete
fermentation, the alcohol is removed by distillation and the
remaining fermentation residues are dried.
[0040] Stillage is the product which remains after the mash has
been converted to sugar, fermented and distilled into ethanol.
Stillage can be separated into two fractions, such as, by
centrifugation or screening: (1) wet cake (solid phase) and (2) the
thin stillage (supernatant). The solid fraction or distillers' wet
grain (DWG) can be pressed to remove excess moisture and then dried
to produce distillers' dried grains (DDG). After ethanol has been
removed from the liquid fraction, the remaining liquid can be
evaporated to concentrate the soluble material into condensed
distillers' solubles (DS) or dried and ground to create distillers'
dried solubles (DDS). DDS is often mixed with DDG to form
distillers' dried grain with solubles (DDGS). DDG, DDGS, and DWG
are collectively referred to as distillers' grain(s).
[0041] In one embodiment of the present disclosure enzymes were
added during and/or preferably after the fermentation in the
production process to the fermented mash and before the separation
step like distillation, where the desired fermentation main product
is separated from the rest of the fermented mash. The enzymes
according to the present disclosure were capable of degrading
components in the fermented mash (beer or beer mash) which improves
the quality of by-products or residues like brewer's grains, dried
distiller's grains, dried distiller's solubles, distiller's dried
grains with solubles, WDG or/and residues of the cereal processing
industry, or mixtures thereof. Components of the fermented mash can
be cell walls, cell-walls of fermenting microorganisms,
microorganisms, fibers etc.
[0042] Surprisingly, the degradation of the fermenting
microorganisms itself by adding the enzyme composition according to
the present disclosure, in particular by using the beta 1,3
glucanase and/or the beta 1,6 glucanase, particularly in
combination with a xylanase and/or a mannanase, results in an
increase of the nutrition content in the beer mash which results in
an improvement of the nutrition quality like the protein content of
the byproducts, as well as reduction of NSPs resulting from the
fermentative organism cell wall like the yeast cell wall.
[0043] Therefore, in advantageous embodiments, the enzyme
compositions used in the methods according to the present
disclosure are capable to degrade the cell wall components of the
fermenting organisms after the fermentation step as well as the
fibers in the beer.
[0044] In one aspect of the present disclosure, the quality of
by-products from a fermentative production process like DDG, DDGS
or WDG can be improved with the methods according to the present
disclosure by reducing the fiber content of the by-products.
[0045] Another aspect of the present disclosure is a better
dewatering of the whole stillage from the fermentative production
process to improve the drying conditions of the by-products.
[0046] Yet another aspect of the present disclosure is the improved
evaporation of water from the thin stillage to improve the
production and composition of the thick stillage or syrup.
[0047] In another aspect of the present disclosure, the amount of
recoverable oil is increased. DDGS following an ethanol production
process from corn typically contains about 13% oil, 31% protein and
56% carbohydrates and other components. Removal of some of the oil
from the DDGS will improve the quality of the DDGS for the feed
market as many feed producers prefer less oil and fat in the DDGS
to make high quality feed.
[0048] The method of the invention may be used on beer derived from
production of any suitable fermentation product. The feedstock for
producing the fermentation product may be any starch- and/or sugar
containing material, preferably starch- and/or sugar containing
plant material, including: sugar cane, tubers, roots, whole grain;
and any combination thereof.
[0049] The starch-containing material may be obtained from cereals.
Suitable starch-containing material includes corn (maize), wheat,
barley, cassava, sorghum, rye, triticale, potato, or any
combination thereof.
[0050] Corn is the preferred feedstock, especially when the
fermentation product is ethanol. The starch-containing material may
also consist of or comprise, e.g., a side stream from starch
processing, e.g., C6 carbohydrate containing process streams that
may not be suited for production of syrups. Beer components include
fiber, hull, germ, oil and protein components from the
starch-containing feedstock as well as non-fermented starch,
yeasts, yeast cell walls and residuals. Production of a
fermentation product is typically divided into the following main
process stages: a) Reducing the particle size of starch-containing
material, e.g., by dry or wet milling; b) Cooking the
starch-containing material in aqueous slurry to gelatinize the
starch, c) Liquefying the gelatinized starch-containing material in
order to break down the starch (by hydrolysis) into maltodextrins
(dextrins); d) Saccharifying the maltodextrins (dextrins) to
produce low molecular sugars (e.g., DP1-2) that can be metabolized
by a fermenting organism; e) Fermenting the saccharified material
using a suitable fermenting organism directly or indirectly
converting low molecular sugars into the desired fermentation
product; f) Recovering the fermentation product, e.g., by
distillation in order to separate the fermentation product from the
fermentation mash.
[0051] As mentioned above beer (or fermented mash) is the
fermentation product consisting of ethanol, other liquids and
solids of a desired fermentation product. According to the
invention the fermentation product may be any fermentation product,
including alcohols (e.g., ethanol, methanol, butanol,
1,3-propanediol); organic acids (e.g., citric acid, acetic acid,
itaconic acid, lactic acid, gluconic acid, gluconate, succinic
acid, 2,5-diketo-D-gluconic acid); ketones (e.g., acetone); amino
acids (e.g., glutamic acid); gases (e.g., H, and CO.sub.2), and
more complex compounds, including, for example, antibiotics (e.g.,
penicillin and tetracycline); enzymes; vitamins (e.g., riboflavin,
B12, beta-carotene); and hormones. Fermentation is also commonly
used in the production of consumable alcohol (e.g., spirits, beer
and wine), dairy (e.g., in the production of yogurt and cheese),
leather, and tobacco industries. In a preferred embodiment the
fermentation product is a liquid, preferably an alcohol, especially
ethanol. The beer contemplated according to the invention may be
the product resulting from a fermentation product production
process including above mentioned steps a) to f). However, the beer
may also be the product resulting from other fermentation product
production processes based on starch- and/or lignocellulose
containing starting material.
[0052] The fermenting organism may be a fungal organism, such as
yeast, or bacteria. Suitable bacteria may e.g. be Zymomonas
species, such as Zymomonas mobilis and E. coli. Examples of
filamentous fungi include strains of Penicillium species. Preferred
organisms for ethanol production are yeasts, such as e.g. Pichia or
Saccharomyces. Preferred yeasts according to the disclosure are
Saccharomyces species, in particular Saccharomyces cerevisiae or
baker's yeast.
[0053] In a further embodiment, the solids from the fermentation
step can be fractionated. After fermentation large pieces of fibers
could be removed prior or after distillation. Removal can be
effected with a surface skimmer before to distillation of beer. The
material can be separated from the ethanol/water mix by, e.g.
centrifugation. Alternatively, fibers and germs can be removed by
screening the whole stillage after distillation or the grinded
grains before fermentation. After germs and large pieces of fibers
are removed the remaining beer or whole stillage are treated with
enzymes or enzyme combinations to further improve the nutritional
quality of the final byproduct like DDGS to be used.
[0054] Based on whole stillage or WDG in one embodiment of the
present disclosure an additional fermenter can be introduced for an
onsite enzyme production, producing a plant and process specific
enzyme mixture with filamentous fungi. The supernatant of this
fermentation comprising enzymes is directly transferred into the
main fermenter or beer well in order to reduce the fiber content
and improve quality and nutritional factors of DDGS, WDG and/or
other by products.
[0055] Furthermore, the use of the enzyme compositions according to
the present disclosure in the beer mash after the fermentation and
before the distillation process can reduce the viscosity of the
beer mash through the degradation of fibers and/or the fermentative
microorganisms in the beer. The reduction of the fibers in the beer
results in a reduction of the fiber content in the by-products. The
early degradation of the NSP's has a direct influence on the
separation and the drying conditions of the by-products like DDGS
in the production process. The lower viscosity results in lower
drying temperatures and also in a shorter drying time resulting in
an improved quality of the by-products. For example, the
temperature sensitive products like proteins and amino acids are
not destroyed.
[0056] Further, by adding the enzymes according to the present
disclosure to the fermented mash before the distillation step is an
advantage since the enzymes in the enzyme compositions are
inactivated during the distillation.
[0057] Due to the improved quality, the by-products and residues
can be used as high quality animal feed with a low fiber and oil
and high protein content.
[0058] In one embodiment, the disclosure relates to methods for
formulating nutritionally useful feed additives as co-products of
the above-referenced methods for improving nutritional
characteristics of a fibrous food product.
[0059] The processes for producing fermentation products includes
the production of a large number of fermentation products
comprising but not limited to alcohols (in particular ethanol);
acids, such as citric acid, itaconic acid, lactic acid, gluconic
acid, lysine; ketones; amino acids, such as glutamic acid, but also
more complex compounds such as antibiotics, such as penicillin,
tetracyclin; enzymes; vitamins, such as riboflavin, B12,
beta-carotene; hormones, such as insulin. Preferred is drinkable
ethanol as well as industrial and fuel ethanol.
[0060] Processes for producing fermentation products, such as
ethanol, from a starch or lignocellulose containing material are
well known in the art. The preparation of the starch-containing
material such as corn for utilization in such fermentation
processes typically begins with grinding the corn in a dry-grind or
wet-milling process. Wet-milling processes involve fractionating
the corn into different components where only the starch fraction
enters into the fermentation process. Dry-grind processes involve
grinding the corn kernels into meal and mixing the meal with water
and enzymes. Generally two different kinds of dry-grind processes
are used. The most commonly used process, often referred to as a
"conventional process," includes grinding the starch-containing
material and then liquefying gelatinized starch at a high
temperature using typically a bacterial alpha-amylase, followed by
simultaneous saccharification and fermentation (SSF) carried out in
the presence of a glucoamylase and a fermentation organism. Another
well-known process, often referred to as a "raw starch hydrolysis"
process (RSH process), includes grinding the starch-containing
material and then simultaneously saccharifying and fermenting
granular starch below the initial gelatinization temperature
typically in the presence of an acid fungal alpha-amylase and a
glucoamylase.
[0061] In a process for producing ethanol from corn, following SSF
or the RSH process the ethanol is distilled from the whole mash
after fermentation. The resulting ethanol-free slurry, usually
referred to as whole stillage, is separated into solid and liquid
fractions (i.e., wet cake and thin stillage containing about 35 and
7% solids, respectively). The thin stillage is often condensed by
evaporation into a thick stillage or syrup and recombined with the
wet cake and further dried into distillers' dried grains with
solubles distillers' dried grain with solubles (DDGS) for use in
animal feed.
[0062] Enzymes used for degrading beer components include
carbohydrases such as alpha-amylase, glucoamylase, cellulase and/or
hemicellulases, such as mannanases, xylanases and beta-glucanases,
pectinases and proteases, or a mixture thereof.
[0063] In advantageous embodiment, the enzyme compositions comprise
a beta-1,3-glucanase, in particular for the degradation of the cell
walls from the fermenting microorganisms. To avoid the degradation
of the fermentative microorganisms the enzyme composition is added
after the fermentation step. As used herein "after the
fermentation" or "after the fermentation step" means that a large
part or all of the fermentable sugars like glucose are converted to
the desired fermentation products such as ethanol.
[0064] In an embodiment, the enzyme composition comprises a
beta-1,3-glucanase and a 1,6-beta-glucanase. In another embodiment,
the enzyme composition comprises a xylanase. In an advantageous
embodiment, the enzyme composition comprises a beta-1,3-glucanase
and a xylanase. In another embodiment, the enzyme composition
comprises a beta-1,3-glucanase, a 1,6-beta-glucanase and a
xylanase.
[0065] In further embodiments, the enzyme composition comprises in
addition a pectinase and/or a protease. In an example the enzyme
composition comprises a beta-1,3-glucanase, a xylanase and a
protease. In another example the enzyme composition comprises a
beta-1,3-glucanase, a xylanase and a pectinase.
[0066] In a further embodiment, enzyme composition comprises a
mannanase. In an advantageous embodiment the enzyme composition
comprises a mannanase and a beta-1,3-glucanase.
[0067] The cell walls of most true fungal and yeast microorganisms
contain a network of glucan, which gives the cell wall strength.
Further major fungal cell walls constituents are mannoprotein and
chitin. For the degradation of the fermented mash components, in
particular for the degradation of the fibers and the fermenting
microorganisms the following enzymes may be used.
[0068] Beta-1,3-glucanases as used herein are enzymes capable of
degrading of glucan. Glucan and chitin are far more resistant to
microbial degradation than cellulose, which is the major
constituent of the cell wall of many yeasts and fungi-like
organisms. Glucan is predominantly beta-1,3-linked with some
branching via 1,6-linkage (Manners et al., Biotechnol. Bioeng, 38,
p. 977, 1973), and is known to be degradable by certain
beta-1,3-glucanase systems, beta-1,3-glucanase includes the group
of endo-beta-1,3-glucanases also called laminarinases (E.C.
3.2.1.39 and E.C. 3.2.1.6, Enzyme Nomenclature, Academic Press,
Inc. 1992).
[0069] A number of beta-1,3-glucanase genes and uses thereof have
been disclosed in the prior art. An example is DD 226012 (Akad.
Wissenshaft, DDR) which concerns a method for production of a
Bacillus beta-1,3-glucanase. Further, JP 61040792 A (DOI K)
describes a cell wall-cytolase beta-1,3-glucanase recombinant
plasmid for removing the cell walls of yeast. The gene is derived
from Arthrobacter and is transformed in Escherichia group bacteria.
EP 440.304 concerns plants provided with improved resistance
against pathogenic fungi transformed with at least one gene
encoding an intracellular chitinase, or in intra- or extracellular
beta-1,3-glucanase. The matching recombinant polynucleotides is
also disclosed. WO 87/01388 (The Trustees of Columbia University)
describes a method for preparing cell lytic enzymes, such as
beta-1,3-glucanases, which can be produced by Oerksovia. WO
92/03557 (Majesty (Her) in Right of Canada) discloses a recombinant
DNA expression vector comprising a 2.7 kb DNA sequence, derived
from Oerskovia xanthineolytica, encoding a beta-1,3-glucanase. From
WO 92/16632 a recombinant DNA sequence coding for a novel protein
with beta-1,3-glucanase activity, is known.
[0070] Examples for commercial available beta-1,3-glucanase are
Rohalase BX from AB Enzymes and Rapidase Glucalees from DSM.
[0071] Hemicellulases as used herein are enzymes capable to break
down hemicellulose. Any hemicellulase suitable for use in
hydrolyzing hemicellulose, may be used. Preferred hemicellulases
include acetylxylan esterases, endo-arabinases, exo-arabinases,
arabinofuranosidases, feruloyl esterase, endo-galactanases,
exo-galactanases, glucuronidases, mannases, xylanases, and mixtures
of two or more thereof. Preferably, the hemicellulase for use in
the present invention is an endo-acting hemicellulase, and more
preferably, the hemicellulase is an exo-acting hemicellulase which
has the ability to hydrolyze hemicellulose under acidic conditions
of below pH 7, preferably pH 3-7.
[0072] In one aspect, the hemicellulase(s) comprises a commercial
hemicellulolytic enzyme preparation. Examples of commercial
hemicellulolytic enzyme preparations suitable for use in the
present invention include, for example, SHEARZYME.TM. (Novozymes
A/S), CELLIC.TM. HTec (Novozymes A/S), CELLIC.TM. HTec2 (Novozymes
A/S), VISCOZYME.RTM. (Novozymes A/S), ULTRAFLO.RTM. (Novozymes
A/S), PULPZYME.RTM. HC (Novozymes A/S), MULTIFECT.RTM. Xylanase
(Genencor), ACCELLERASE.RTM. XY (Genencor), ACCELLERASE.RTM. XC
(Genencor), ECOPULP.RTM. TX-200A (AB Enzymes), HSP 6000 Xylanase
(DSM), DEPOL.TM. 333P (Biocatalysts Limit, Wales, UK), DEPOL.TM.
740L. (Biocatalysts Limit, Wales, UK), and DEPOL.TM. 762P
(Biocatalysts Limit, Wales, UK).
[0073] Preferably, the hemicellulase for use in the present
disclosure is an endo-acting hemicellulase, which has the ability
to hydrolyze hemicellulose under acidic conditions of below pH 7.
An example of hemicellulase suitable for use in the present
invention includes VISCOZYME L.TM. (available from Novozymes A/S,
Denmark), Rohament GMP.TM. (available from AB Enzymes).
[0074] In an embodiment the hemicellulase is a xylanase. In an
embodiment the xylanase may preferably be of microbial origin, such
as of fungal origin (e.g., Aspergillus, Fusarium, Humicola,
Meripilus, Trichoderma) or from a bacterium (e.g., Bacillus). In a
preferred embodiment the xylanase is derived from a filamentous
fungus, preferably derived from a strain of Aspergillus, such as
Aspergillus aculeatus; or a strain of Humicola, preferably Humicola
lanuginosa. Examples of xylanases useful in the methods of the
present invention include, but are not limited to, Aspergillus
aculeatus xylanase (GeneSeqP:AAR63790; WO 94/21785), Aspergillus
fumigatus xylanases (WO 2006/078256), and Thielavia terrestris NRRL
8126 xylanases (WO 2009/079210). The xylanase may preferably be an
endo-1,4-beta-xylanase, more preferably an endo-1,4-beta-xylanase
of GH 10 or GH 1 1. Examples of commercial xylanases include
SHEARZYME.TM., BIOFEED WHEAT.TM., HTec and HTec2 from Novozymes
A/S, Denmark.
[0075] Examples of beta-xylosidases useful in the methods of the
present invention include, but are not limited to, Trichoderma
reesei beta-xylosidase (UniProtKB/TrEMBL accession number Q92458),
Talaromyces emersonii (SwissProt accession number Q8X212), and
Neurospora crassa (SwissProt accession number Q7SOW4).
[0076] According to the invention beer may in step i) be subjected
to an effective amount of any xylanase (EC 3.2.1.8), such as any of
below mentioned xylanases. Xylanase activity may be derived from
any suitable organism, including fungal and bacterial organisms.
Fungal xylanases may be derived from strains of genera including
Aspergillus, Disporotrichum, Penicillium, Neurospora, Fusarium and
Trichoderma.
[0077] Examples of suitable bacterial xylanases include xylanases
derived from a strain of Bacillus, such as Bacillus subtilis, such
as the one disclosed in U.S. Pat. No. 5,306,633 or
[0078] Contemplated commercially available xylanases include
SHEARZYM E.TM., BIOFEED WHEAT.TM., (from Novozymes AJS), Econase
CE.TM. (from AB Enzymes), Depol 676.TM. (from Biocatalysts Ltd.)
and SPEZYME.TM. CP (from Genencor Int.).).
[0079] Xylanase may be added in an amount effective in the range
from 0.16.times.10<6>-460.times.10<6> Units per ton
beer mash.
[0080] Mannanases are hemicellulases classified as EC 3.2.1.78, and
called endo-1,4-beta-mannosidase. Mannanase includes
beta-mannanase, endo-1,4-mannanase, and galacto-mannanase.
Mannanase is preferably capable of catalyzing the hydrolysis of
1,4-beta-D-mannosidic linkages in mannans, including glucomannans,
galactomannans and galactoglu-comannans. Mannans are
polysaccharides primarily or entirely composed of D-mannose units.
The mannanase may be of any origin such as a bacterium or a fungal
organism. In a specific embodiment the mannanase is derived from a
strain of the filamentous fungus genus Trichoderma, preferably
Trichoderma reseei. In an embodiment the mannanase is one of the
mannanases described in WO2008/009673.
[0081] Mannanases have been identified in several Bacillus
organisms. For example, Talbot et al., Appl. Environ. Microbiol.,
Vol. 56, No. 11, pp. 3505-3510 (1990) describes a beta-mannanase
derived from Bacillus stearothermophilus. Mendoza et al., World J.
Microbiol. Biotech., Vol. 10, No. 5, pp. 551-555 (1994) describes a
beta-mannanase derived from Bacillus subtilis. JP-A-03047076
discloses a beta-mannanase derived from Bacillus sp. JP-A-63056289
describes the production of an alkaline, thermo stable
beta-mannanase. JP-A-63036775 relates to the Bacillus microorganism
FERM P-8856 which produces beta-mannanase and beta-mannosidase.
JP-A-08051975 discloses alkaline beta-mannanases from alkalophilic
Bacillus sp. AM-001. A purified mannanase from Bacillus
amyloliquefaciens is disclosed in WO 97/11164. WO 91/18974
describes a hemicellulase such as a glucanase, xylanase or
mannanase active. Examples of commercially available mannanases
include GAMANASE.TM. available from Novozymes A/S Denmark and
Rohapect GMP.TM. available from AB Enzymes GmbH.
[0082] Mannanase may be added in an amount effective in the range
from 0.3.times.10<6>-1.6.times.10<6> Units per ton beer
mash.
[0083] A cellulase, used in accordance with the disclosure, may be
any cellulase, in particular of microbial origin, in particular
fungal or bacterial origin such as a cellulase derivable from a
strain of a filamentous fungus (e.g., Aspergillus, Trichoderma,
Humicola, Fusarium). Preferably, the cellulase acts on both
cellulosic and lignocellulosic material. Preferred cellulases for
use in the present invention include endo-acting cellulases,
exo-acting celluases and cellobiases, and combinations thereof.
Examples of commercially available cellulases suitable according to
the present invention include, for example, CELLULCLAST.TM.
(available from Novozymes A/S), LAMINEX.TM. and SPEZYME.TM. CP
(Genencor Int.) and Econase CE.TM. (from AB Enzymes GmbH), Rohalase
BX.TM. (from Ab Enzymes GmbH), Cellulase 13P.TM. (from Biocatalysts
Ltd.). Cellulase may be added in amounts effective in the range or
from 0.03.times.10<6>-16.times.10<6> Units per ton
substrate (in beer mash)
[0084] The pectinase used in the methods according to the present
disclosure may be any pectinase, in particular of microbial origin,
in particular of bacterial origin, such as a pectinase derived from
a species within the genera Bacillus, Clostridium, Pseudomonas,
Xanthomonas and Erwinia, or of fungal origin, such as a pectinase
derived from a species within the genera Trichoderma or
Aspergillus, in particular from a strain within the species A.
niger and A. aculeatus. Contemplated commercially available
pectinases include Pectinex Ultra-SPL.TM. (from Novozymes),
Pectinex Ultra Color (from Novozymes), Rohapect Classic (from AB
Enzymes), Rohapect 10L (from AB Enzymes). Pectinase may be added in
an amount effective in the range from
1.4.times.10<9>-23500.times.10<9> Units per ton beer
mash.
[0085] Proteases as used in the present disclosure are enzymes that
catalyze the cleavage of peptide bonds. Suitable proteases include
fungal and bacterial proteases. Preferred proteases are acidic
proteases, i.e., proteases characterized by the ability to
hydrolyze proteins under acidic conditions below pH 7
[0086] Suitable acid fungal proteases include fungal proteases
derived from Aspergillus, Mucor, Rhizopus, Candida, Coriolus,
Endothia, Enthomophtra, Irpex, Penicillium, Sclerotium and
Toru-lopsis. Commercial proteases include GC 106.TM. and
SPEZYME.TM. FAN (available from Genencor, USA). Suitable bacterial
proteases, although not acidic proteases, include the commercially
available products ALCALASE.TM. and NEUTRASE.TM. (available from
Novozymes A/S).
[0087] Protease may be added in an amount effective in the range
from 0.002.times.10<6>-314.times.10<6> Units per ton
beer mash.
[0088] Any phytase may be used in the methods of the present
disclosure. Phytases are enzymes that degrade phytates and/or
phytic acid by specifically hydrolyzing the ester link between
inositol and phosphorus. Phytase activity is credited with
phosphorus and ion availability in many ingredients. In some
embodiments, the phytase is capable of liberating at least one
inorganic phosphate from an inositol hexaphosphate (e.g., phytic
acid). Phytases can be grouped according to their preference for a
specific position of the phosphate ester group on the phytate
molecule at which hydrolysis is initiated (e.g., 3-phytase (EC
3.1.3.8) or 6-phytase (EC 3.1.3.26)). An example of phytase is
myo-inositol-hexakiphosphate-3-phosphohydrolase. Phytases can be
obtained from microorganisms such as fungal and bacterial
organisms. For example, the phytase may be obtained from
filamentous fungi such as Aspergillus (e.g., A. ficuum, A.
fumigatus, A. niger, and A. terreus), Cladospirum, Mucor (e.g.,
Mucor piriformis), Myceliop tora (e.g., M. termopila), Penicillium
(e.g., P. ordei (ATCC No. 22053)), P. piceum (ATCC No. 10519), or
P. brevi-compactum (ATCC No. 48944), Talaromyces (e.g., T.
thermophilus), Thermomyces (WO 99/49740), and Trichoderma spp.
(e.g., T. reesei). In an embodiment, the phytase is derived from
Buttiauxiella spp. such as B. agrestis, B. brennerae, B.
ferragutiase, B. gaviniae, B. izardii, B. noackiae, and B.
warmboldiae. In some embodiments, the phytase is a phytase
disclosed in WO 2006/043178.
[0089] Example 1 shows the enzymatic dewatering of the whole
stillage after treating the beer mash with enzymes capable of
degrading at least one beer mash component. Therefore, one aspect
of the disclosure relates to a method to improve the quality of
WDG, DDG and/or DDGS comprising the steps of: i) subjecting beer to
one or more enzymes capable of degrading one or more beer
components, ii) distillation, iii) separating the material into a
solid fraction and a liquid fraction. The solid fraction is often
referred to as "wet cake" and the liquid fraction is often referred
to as "thin stillage".
[0090] In accordance with the purposes of the present invention as
described herein, in one aspect of the present disclosure a method
is provided for improving the nutritional quality of a by-product
or residue of a fermentative production process, comprising
inoculating the by-product or residue with at least one filamentous
fungus, fermenting the by-product or residue and separating at
least one enzyme from the fermented by-product or residue; and
providing the enzyme to fermented mash (beer mash) of an
fermentative production process, preferably an ethanol production
process. The filamentous fungus may be selected from the group
consisting of Rhizopus, Aspergillus, Trichoderma, and any
combination thereof. The by-product or residue is preferably a
fibrous by-product and may be selected from the group consisting of
spent brewer's grains, dried distiller's grains, dried distiller's
solubles, distiller's dried grains with solubles and WDG, and
mixtures thereof.
[0091] In another aspect the present disclosure relates to methods
of producing ethanol from starch containing material, said method
comprising the steps of: [0092] v) Converting starch-containing
material to fermentable sugars [0093] vi) Fermentation of the
fermentable sugars with a microorganism to fermented mash [0094]
vii) Subjecting the fermented mash after the fermentation process
to an enzyme composition comprising an enzyme or a mixture of
enzymes [0095] viii) Separation of the ethanol in the fermented
mash by distillation
[0096] Converting starch-containing material to fermentable sugars
can be done by (a) liquefying a starch-containing material and (b)
saccharifying the liquefied material obtained in step (a).
[0097] The liquefaction is preferably carried out in the presence
of an alpha-amylase, preferably a bacterial alpha-amylase or acid
fungal alpha-amylase. In an embodiment, a pullulanase, isoamylase,
and and/or phytase is added during liquefaction.
[0098] Preferred organisms for ethanol production are yeasts, such
as e.g. Pichia or Saccharomyces. Preferred yeast according to the
disclosure is Saccharomyces species, in particular Saccharomyces
cerevisiae or baker's yeast. The yeast cells may be added in
amounts of 10.sup.5 to 10.sup.12, preferably from 10.sup.7 to
10.sup.10, especially 5.times.10.sup.7 viable yeast count per ml of
fermentation broth. During the ethanol producing phase the yeast
cell count should preferably be in the range from 10.sup.7 to
10.sup.10, especially around 2.times.10.sup.8. Further guidance in
respect of using yeast for fermentation can be found in, e.g., "The
alcohol Textbook" (Editors K. Jacques, T. P. Lyons and D. R.
Kelsall, Nottingham University Press, United Kingdom 1999), which
is hereby incorporated by reference
[0099] The microorganism used for the fermentation is added to the
mash and the fermentation is ongoing until the desired amount of
fermentation product is produced; in a preferred embodiment wherein
the fermentation product is ethanol to be recovered this may, e.g.
be for 24-96 hours, such as 35-60 hours. The temperature and pH
during fermentation is at a temperature and pH suitable for the
microorganism in question and with regard to the intended use of
the fermentation product, such as, e.g., in an embodiment wherein
the fermenting organism is yeast and the product is ethanol for
recovery the preferred temperature is in the range about 26-34 C,
e.g. about 32 C, and at a pH e.g. in the range about pH 3-6, e.g.
about pH 4-5.
[0100] In another embodiment wherein the fermenting organism is
yeast, and the fermented mash is to be used as a beer, the
temperature of the mash the preferred temperature is around 12-16
C, such around 14 C.
[0101] As mentioned above, the fermenting organism is preferably
yeast, e.g., a strain of Saccharomyces cerevisiae or Saccharomyces
diastaticus. In an advantageous embodiment a yeast strain of
Saccharomyces diastaticus is used (SIHA Amyloferm.RTM., E. Begerow
GmbH&Co, Langenlonsheim, Germany) since their exo-amylase
activity can split liquid starch and also dextrin, maltose and
melibiose.
[0102] In the liquefaction step the gelatinized starch (downstream
mash) is broken down (hydrolyzed) into maltodextrins (dextrins). To
achieve starch hydrolysis a suitable enzyme, preferably an
alpha-amylase, is added. 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 an alpha-amylase
may be 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 about 1-15 minutes, preferably
for about 3-10 minutes, especially around about 5 minutes. The
slurry is cooled to 60-95.degree. C. and more alpha-amylase may be
added to complete the hydrolysis (secondary liquefaction). The
liquefaction process is usually carried out at a pH of 4.0 to 6.5,
in particular at a pH of 4.5 to 6.
[0103] The saccharification step and the fermentation step may be
performed as separate process steps or as a simultaneous
saccharification and fermentation (SSF) step. The saccharification
is carried out in the presence of a saccharifying enzyme, e.g. a
glucoamylase, a beta-amylase or maltogenic amylase. Optionally a
phytase and/or a protease is added.
[0104] Saccharification may be carried out using conditions well
known in the art with a saccharifying enzyme, e.g., beta-amylase,
glucoamylase or maltogenic amylase, and optionally a debranching
enzyme, such as an isoamylase or a pullulanase. For instance, a
full saccharification process may last up to from about 24 to about
72 hours, however, it is common to do a pre-saccharification for
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 a temperature from
20-75.degree. C., preferably from 40-70.degree. C., typically
around 60.degree. C., and at a pH between 4 and 5, normally at
about pH 4.5.
[0105] The most widely used process to produce a fermentation
product, especially ethanol, is the simultaneous saccharification
and fermentation (SSF) process, in which there is no holding stage
for the saccharification, meaning that a fermenting organism, such
as a yeast, and enzyme(s), including the hemicellulase(s) and/or
specific endoglucanase(s), may be added together. SSF is typically
carried out at a temperature from 25.degree. C. to 40.degree. C.,
such as from 28.degree. C. to 35.degree. C., from 30.degree. C. to
34.degree. C., preferably around about 32.degree. C. In an
embodiment, fermentation is ongoing for 6 to 120 hours, in
particular 24 to 96 hours.
[0106] After the fermentation, the fermented mash is subjected to
an enzyme composition according to the present disclosure. In an
embodiment, the enzyme composition comprises a beta-1,3-glucanase.
In another embodiment the enzyme composition comprises a
beta-1,3-glucanase and a 1,6-beta-glucanase. In another embodiment,
the enzyme composition comprises a xylanase. In an advantageous
embodiment, the enzyme composition comprises a beta-1,3-glucanase
and a xylanase. In another embodiment, the enzyme composition
comprises a beta-1,3-glucanase, a 1,6-beta-glucanase and a
xylanase. In further embodiments, the enzyme composition comprises
in addition a pectinase and/or a protease. In an example the enzyme
composition comprises a beta-1,3-glucanase, a xylanase and a
protease. In another example the enzyme composition comprises a
beta-1,3-glucanase, a xylanase and a pectinase. In a further
embodiment, enzyme composition comprises a mannanase. In an
advantageous embodiment the enzyme composition comprises a
mannanase and a beta-1,3-glucanase.
[0107] In a particular embodiment, the process of the invention
further comprises, prior to liquefying the starch-containing
material the steps of: [0108] reducing the particle size of the
starch-containing material, preferably by milling; and [0109]
forming a slurry comprising the starch-containing material and
water.
[0110] The aqueous slurry may contain from 10-55 w/w % dry solids
(DS), preferably 25-45 w/w % dry solids (DS), more preferably 30-40
w/w % dry solids (DS) of the starch-containing material. The slurry
is heated to above the gelatinization temperature and an
alpha-amylase, preferably a bacterial and/or acid fungal
alpha-amylase, may be added to initiate liquefaction (thinning).
The slurry may be jet-cooked to further gelatinize the slurry
before being subjected to an alpha-amylase in step (a).
[0111] In a preferred embodiment, the starch containing material is
milled cereals, preferably barley or corn, and the methods comprise
a step of milling the cereals before step (a). In other words, the
disclosure also encompasses methods, wherein the starch containing
material is obtainable by a process comprising milling of cereals,
preferably dry milling, e.g. by hammer or roller mils. Grinding is
also understood as milling, as is any process suitable for opening
the individual grains and exposing the endosperm for further
processing. Two processes of milling are normally used in alcohol
production: wet and dry milling. The term "dry milling" denotes
milling of the whole grain. In dry milling the whole kernel is
milled and used in the remaining part of the process Mash
formation. The mash may be provided by forming a slurry comprising
the milled starch containing material and brewing water. The
brewing water may be heated to a suitable temperature prior to
being combined with the milled starch containing material in order
to achieve a mash temperature of 45 to 70.degree. C., preferably of
53 to 66.degree. C., more preferably of 55 to 60.degree. C. The
mash is typically formed in a tank known as the slurry tank.
[0112] Subsequent to fermentation the fermentation product may be
separated from the fermentation medium. The slurry may be distilled
to extract the desired fermentation product or the desired
fermentation product from the fermentation medium by micro or
membrane filtration techniques. Alternatively the fermentation
product may be recovered by stripping. Methods for recovering
fermentation products are well known in the art. Typically, the
fermentation product, e.g., ethanol, with a purity of up to, e.g.,
about 96 vol. % ethanol is obtained.
[0113] Following the completion of the fermentation process, the
material remaining is considered the whole stillage. As used
herein, the term "whole stillage" includes the material that
remains at the end of the fermentation process both before and
after recovery of the fermentation product, e.g., ethanol. The
fermentation product can optionally be recovered by any method
known in the art. In one embodiment, the whole stillage is
separated or partitioned into a solid and liquid phase by one or
more methods for separating the thin stillage from the wet cake.
Such methods include, for example, centrifugation and decanting.
The fermentation product can be optionally recovered before or
after the whole stillage is separated into a solid and liquid
phase.
[0114] Thus, in one embodiment, the methods of the disclosure
further comprise distillation to obtain the fermentation product,
e.g., ethanol. The fermentation and the distillation may be carried
out simultaneously and/or separately/sequentially; optionally
followed by one or more process steps for further refinement of the
fermentation product.
[0115] In an embodiment, the aqueous by-product (whole stillage)
from the distillation process is separated into two fractions,
e.g., by centrifugation: wet grain (solid phase), and thin stillage
(supernatant). In another embodiment, the methods of the disclosure
further comprise separation of the whole stillage produced by
distillation into wet grain and thin stillage; and recycling thin
stillage to the starch containing material prior to liquefaction.
In one embodiment, the thin stillage is recycled to the milled
whole grain slurry. The wet grain fraction may be dried, typically
in a drum dryer. The dried product is referred to as distillers
dried grains, and can be used as mentioned above as high quality
animal feed. The thin stillage fraction may be evaporated providing
two fractions (see FIG. 1 and FIG. 2), (i) a condensate fraction of
4-6% DS (mainly of starch, proteins, oil and cell wall components),
and (ii) a syrup fraction, mainly consisting of limit dextrins and
non-fermentable sugars, which may be introduced into a dryer
together with the wet grains (from the whole stillage separation
step) to provide a product referred to as distillers dried grain
with solubles, which also can be used as animal feed. Thin stillage
is the term used for the supernatant of the centrifugation of the
whole stillage. Typically, the thin stillage contains 4-6% DS
(mainly starch and proteins) and has a temperature of about
60-90.degree. C. In another embodiment, the thin stillage is not
recycled, but the condensate stream of evaporated thin stillage is
recycled to the slurry containing the milled whole grain to be jet
cooked.
[0116] Further details on how to carry out liquefaction,
saccharification, fermentation, distillation, and recovering of
ethanol are well known to the skilled person.
[0117] Methods for dewatering stillage and for extracting oil from
a fermentation product are known in the art. These methods include
decanting or otherwise separating the whole stillage into wet cake
and thin stillage. See, e.g., U.S. Pat. Nos. 6,433,146, 7,601,858,
and 7,608,729, and U.S. Application Publication No. 2010/0058649.
Furthermore, the thin stillage can be evaporated or condensed into
syrup or thick stillage from which the oil can be extracted
utilizing centrifugation, filtering, heat, high temperature,
increased pressure, or a combination of the same. Another way to
extract oil is to lower the pH of the thin stillage or syrup. The
use of surfactants to break emulsions also enhances oil extraction.
Presses can also be used for dewatering. In one embodiment of the
disclosure, the presence of beta 1,3-glucanase and/or xylanase in
the fermented mash after the fermentation increases the amount of
oil in the thin stillage and further the syrup or thick
stillage.
[0118] The fermentation product(s) can be optionally recovered from
the fermentation medium using any method known in the art
including, but not limited to, chromatography, electrophoretic
procedures, differential solubility, distillation, or extraction.
For example, alcohol is separated from the fermented cellulosic
material and purified by conventional methods of distillation as
mentioned above. Ethanol with a purity of up to about 96 vol. % can
be obtained, which can be used as, for example, fuel ethanol,
drinking ethanol, i.e., potable neutral spirits, or industrial
ethanol.
[0119] The inventions described and claimed herein are not to be
limited in scope by the specific embodiments herein disclosed,
since these embodiments are intended as illustrations of several
aspects of the invention. Any equivalent embodiments are intended
to be within the scope of this invention. Indeed, various
modifications of the invention in addition to those shown and
described herein will become apparent to those skilled in the art
from the foregoing description. Such modifications are also
intended to fall within the scope of the appended claims. In the
case of conflict, the present disclosure including definitions will
control. Various references are cited herein, the disclosures of
which are incorporated by reference in their entireties. The
present invention is further described by the following examples
which should not be construed as limiting the scope of the
invention.
EXAMPLES
A) Processes for Producing Fermentation Products
[0120] In one embodiment, the process of the production of ethanol
form corn was performed as follows:
a) reducing the particle size of the starch-containing material by
milling: [0121] corn was milled to <2 mm particle size b)
forming a slurry comprising the starch-containing material and
water [0122] 10 kg corn were mixed with tap water at 35.degree. C.
to obtain a .about.31.25% solid solution, total volume 30 liter
[0123] pH range was 5.6-6.0 c) liquefying of the starch-containing
material [0124] temperature was increased to 90.degree. C. [0125] 7
ml alpha-amylase (Novozymes Liquozyme SC) were added [0126]
1.Salinity. antifoam was added (30 ml) [0127] incubation for 90 min
at 90.degree. C. and stirring at 150 rpm d) saccharifying of the
liquefied material obtained [0128] slurry was cooled to 30.degree.
C., pH adjusted to .about.4 with 1M (NH4)2SO4-12 ml Glucoamylase
(Novozymes Spirizyme Ultra) was added e) fermentation [0129] Yeast
propagation: 200 ml YNB-starch medium was incubated overnight
(30.degree. C., 150 rpm) and inoculated with 2 g yeast (SIHA
Amyloferm), the complete pre-culture was added to the fermentation
[0130] addition of 10 g ((NH.sub.4).sub.2SO.sub.4) as nitrogen
source [0131] yeast addition [0132] pH was titrated to 4.0 with
ammoniac solution (25%),--incubation for max. 48 h at 30.degree. C.
and 100 rpm [0133] 2-ml samples were taken every 12 hrs to monitor
fermentation progress (sugar-, ethanol concentration)
B) Enzymes
[0134] The following enzymes listed in Table 1 were used alone or
in different combinations.
TABLE-US-00001 TABLE 1 Typ Activity Shortname Cellulase 565.84 U/g
Cel2 endo-1,4 beta 6538.01 U/g Man3 Mannanase Pectinase 90349.55
U/ml Pec3 .beta. 1-3 Glucanase 10016.97 U/g Glu1 Xylanase 34094.02
U/g Xyl4 Xylanase 35094.00 U/g Xyl7
C) Enzyme Product Activity Determination with DNSA
Substrates:
[0135] endo Glucanase: B-Glucan from barley, low viscosity
(Megazyme) [0136] Xylanase: Xylan from Birchwood (Sigma) [0137]
Mannanase: Galactomannan, carob [0138] Pectinase: Polygalacturonic
acid (Sigma) Substrates were dissolved in buffer to a concentration
of 0.8% (w/v)
Buffer:
[0139] 50 mM NaAcetat, pH 4.5
[0140] The Enzymes were diluted in buffer, the right enzyme
concentration must be determined for each enzyme. A 90 .mu.l
substrate and 10 .mu.l enzyme solution were mixed. A blank was
measured replacing enzyme solution with water. Incubation for 30
min at 37.degree. C. (Reducing sugars were measured after mixing of
50 .mu.l of the incubated substrate--enzyme mix with 50 .mu.l
DNSA-Reagent.
[0141] The activity is calculated as Units per .mu.l or mg of
enzyme product. 1 unit is defined as the amount of formed reducing
ends in .mu.mol per minute.
D) ASSAY on endo-1,3-Beta-Glucanase
[0142] The substrate employed is Azurine-crosslinked pachyman
(AZCL-Pachyman). The substrate is prepared by dyeing and
crosslinking highly purified pachyman to produce a material which
hydrates in water but is water insoluble. Hydrolysis by
endo-1,3-.beta.-glucanase produces water soluble dyed fragments,
and the rate of release of these (increase in absorbance at 590 nm)
can be related directly to enzyme activity. The substrate is
supplied commercially in a ready-to-use tablet form as
1,3-Beta-Glucazyme Tablets (Megazyme International Ireland Ltd,
1,3-BETA-GLUCAZYME TABLETS)
E) Protease Assay
[0143] Protease activity of enzyme products was determined with
TNBS-Assay as described in Example 6.
[0144] One protease unit is defined as the formation of glycin
equivalents per minute. The enzyme activities are shown in Table
2.
TABLE-US-00002 TABLE 2 Main Shortname Activity Unit Cel1 79.64 U/ml
Cel2 565.84 U/g Cel3 1061.00 U/ml Man1 6205.96 U/g Man2 7988.38 U/g
Man3 6538.01 U/g Pec1 28242.70 U/ml Pec2 37473.18 U/ml Pec3
90349.55 U/ml Pec4 117283.35 U/ml Pec5 44082.56 U/ml Glu1 10016.97
U/g Glu2 10594.57 U/g Glu3 3583.18 U/g Glu5 763.00 U/ml Xyl1
2264.56 U/ml Xyl2 4434.68 U/g Xyl3 314.83 U/g Xyl4 34094.02 U/g
Xyl5 4.28 U/g Xyl7 35094.00 U/g Xyl8 14108.00 U/g Xyl9 75000.00 U/g
Xyl10 790.00 U/g Xyl 11 1178.00 U/g Xyl12 16603.50 U/g
Example 1
Dewatering of Whole Stillage
[0145] Beer (8.7 wt-% dry solids, pH=4.0 from conventional,
dry-milled ethanol fermentation was used as substrate.
[0146] An aliquot (50 mL) of whole stillage was placed into a
centrifuge tube and heated to 37<0>C. Total enzyme
concentration added was 200 ppm, the mixture was gently agitated
overnight on a rotary shaker.
[0147] The tube was centrifuged for 5 minutes at 2000 rpm. The
supernatant was decanted and the resulting wet cake was weighed and
compared to the control without addition of any enzyme. The
results, along with several enzymes and enzyme combinations tested,
are shown in Table 2 and Table 3.
TABLE-US-00003 TABLE 2 Enzyme wet cake StdDev Cel1 91.9% 1.15 Cel2
90.8% 1.06 Man1 87.8% 4.01 Man2 86.7% 2.53 Man3 Men1 99.6% 0.4 Pec1
97.8% 2.48 Pec2 87.1% 8.16 Pec3 80.8% 0.71 Pec4 82.0% 0.57 Pec5
94.6% 3.65 Pro1 87.8% 1.12 Pro2 96.2% 0.89 Pro3 93.7% 1.34 Pro4
91.5% 1.97 Pro5 93.3% 4.77 Glu1 85.8% 1.33 Glu2 79.6% 0.81 Glu3
90.3% 0.65 Xyl1 79.4% 1.29 Xyl2 91.3% 1.17 Xyl3 86.1% 1.16 Xyl4
77.0% 0.92 Xyl5 90.1% 0.41 Xyl6 89.6% 2.05
TABLE-US-00004 TABLE 3 Enzyme combinations Wet cake StdDev Xyl4
87.4% 0.23 Xyl4 + Glu1 83.1% 0.26 Xyl4 + Pec3 86.7% 0.63 Xyl4 +
Man3 82.7% 0.30 Xyl4 + Cel2 88.3% 0.05 Xyl4 + Pro1 87.4% 0.58 Xyl4
+ Glu1 + Prot1 78.5% 0.07 Xyl4 + Pec3 + Prot1 82.6% 0.20 Xyl4 +
Man3 + Prot1 82.5% 0.13 Xyl4 + Cel2 + Prot1 82.7% 0.19 Xyl4 + Pec3
+ Man3 82.7% 0.19 Xyl4 + Cel2 + Man3 83.2% 0.11 Xyl4 + Cel2 + Pec3
82.6% 0.13 Xyl4 + Pec3 + Glu1 81.4% 0.04
Example 2
Fiber Reduction in DDGS after Enzymatic Treatment Measured by
Determination of ADF and NDF
[0148] Acid Detergent Fibers (ADF) and Neutral Detergent Fibers
(NDF) are dietary fibers and can be soluble and insoluble. ADF
containing lignin and cellulose, NDF is containing ADF and
hemicelluloses. Soluble fibers are responsible for viscosity
effects within the digestion process and responsible for reduce
energy and protein uptake (cage effect). Reduced ADF/NDF values
leads to better digestion within the intestinal due to the reduced
viscosity in coincidence with better protein and energy
release.
[0149] Beer (10 wt-% dry solids, pH=4.0) from conventional,
dry-milled ethanol fermentation was used as substrate.
[0150] An aliquot (50 mL) of beer was placed into a centrifuge tube
and heated to 37.degree. C. The total enzyme amount added was in
the range of 100 ppm to 400 ppm, the mixture was gently agitated
for 6 hours on a rotary shaker. After thermal enzyme inactivation
(1 hour at 80.degree. C.) the whole stillage was dried and milled.
The dried stillage was analyzed for ADF/NDF according the protocols
of VDLUFA Bd. III, 6.5.2. (Method book III "The chemical analysis
of feedstuff" of VDLUFA 1st-7th supplement delivery)
[0151] The results are shown FIGS. 4 to 7. FIG. 4 shows the result
of adding an enzyme composition comprising beta-1,3-glucanase
[0152] as the main activity (Rohalase BX, AB Enzymes). The measured
parameters are Neutral Detergent Fibers (NDF) and Acid Detergent
Fibers (ADF). The enzyme dosage was 200 g/t of beer. These results
shows clearly that the beta-1,3-glucanase enzyme reduces the values
of ADF and NDF by 9% and 10.6%.
[0153] FIG. 5 shows the effect of an enzyme composition comprising
a xylanase as the main activity (Xylanase 2 XP Conc, Dyadic)
applied in two concentrations of 100 and 200 g/t of beer. The
xylanase reduces the fiber content of DDGS which can be seen in the
clear reduction of ADF and NDF. With an enzymes dosage of 100 g/t
the NDF concentration is only reduced by 1.2% whereas the reduction
was 17.3% for a dosage of 200 g/t.
[0154] FIG. 6 shows the effect of an enzyme composition comprising
as main activities xylanase and 1,3-beta-glucanase activity, added
in concentrations of each 200 g/t beer.
[0155] Compared to FIG. 5 where xylanase used in a concentration of
100 g/t alone showed only minimal effect on fiber reduction, the
combination of xylanase and 1,3-beta-glucanase has a higher effect.
The ADF value is reduced by 2.8% and the NDF value by 8.2%.
[0156] FIG. 7 shows the ADF/NDF reduction of the combination of
xylanase and 1,3-beta-glucanase in two combinations each with
additional enzymes. An enzyme composition comprising in addition
pectinase activity (Rohapect Classic, AB Enzymes) is showing an
additional reduction of NDF (5.8%) but the enzyme is overriding the
effect for ADF. The additional application of proteases (Protease
A, Amano Japan) is affecting the ADF and NDF reduction positive.
The NDF value is reduced again by 3.2%.
Example 3
De-Oiling Improvement
[0157] The oil content of DDGS is sometimes higher than desired and
methods of recovering more oil as a separate by-product for use in
biodiesel production or other biorenewable products are sought.
Much of the work in oil recovery from fermentation processes has
focused on improving the extractability of the oil from the whole
stillage. As mentioned in the specification, a better de-oiling
leads to a better oil separation within the ETOH process. The corn
oil production is a high valuable byproduct for the food and feed
industry also for the Biodiesel production.
[0158] After the reaction of an enzyme composition comprising as
main activities xylanase (200 ppm, Xylanase 2 XP Conc, Dyadic) and
1,3-beta-glucanase activity (200 ppm, Rohalase BX, AB Enzymes),
followed by the centrifugation (conditions: 3000 rpm for 10 min.)
of the whole stillage an improved oil separation in the thin
stillage was observed. FIGS. 8 and 9 are showing results of thin
stillages after centrifugation. The thin stillage where the beer
was treated with the enzyme composition comprising as main
activities xylanase and 1,3-glucanase activity (FIG. 9) shows a
much bigger oil layer on the supernatant compared to the thin
stillage where the beer was not treated with enzymes (FIG. 8). FIG.
8 shows that no oil separation has been adjusted. FIG. 9 shows that
a clear separation of oil, forming a thick oil layer can be
observed.
Example 4
Feeding Tests
[0159] With the following animal trial it was shown that the
exchange of expensive feed ingredients, like soybean meal, corn or
wheat, with DDGS, especially DDGS that is modified during the
production processes and methods according to the present
disclosure, is possible and do not results in lower weight gains.
The goal was to show that DDGS-treated can replace Soybean meal and
wheat without negative effects on animal growth. [0160] quails as
tests animals fed from day 1-day 23 [0161] Replicates 10 cages with
2 animals for each treatment [0162] DDGS inclusion in feed 20%
[0163] positive control was standard wheat and soybean meal diet
without DDGS [0164] All feed ratios were balanced to be isonitrogen
(every feed contains the same amount of CP) and isocaloric (each
feed contains the same amount of metabolized energy) [0165] 3
animal groups with normal feed, untreated DDGS and enzymatically
optimized DDGS [0166] Performance parameters: weight gain, Feed
conversion ration
[0167] For the tests, two Different DDGS types were produced:
a) DDGS resulting from a treatment of the fermented mash in the
ethanol production from A) of the present disclosure with an enzyme
composition comprising a xylanase (200 ppm, Xylanase 2 XP Conc,
Dyadic) and a 1,3-glucanase (200 ppm, Rohalase BX, AB Enzymes)
(DDGS-treated) b) DDGS produced without an enzyme treatment of the
fermented mash (DDGS-blank).
[0168] As a positive control a standard quail feed was used. FIG.
10 shows the results and the weight gain of quails fed with
different feed stuff. In the starter phase (day 0-14) the normal
feed leads to highest weight gain. In the grower phase, feed with
20% of the DDGS-treated, lead to the highest weight gain and
outperformed the other two feeds. Based on the animal trial data,
the feed containing DDGS-treated is showing a clear outperformance.
The DDGS-treated can be included to quail feed at a concentration
of 20% without negative effects on growth performance.
Example 5
De-Watering of Whole Stillage
[0169] As mentioned above in the specification, a better de
watering capability of the whole stillage results in a wet cake
with a higher dry mass. The advantage here is less energy
consumption while drying.
[0170] For that example the de watering capability of the whole
stillage during the centrifugation was tested and determined. Two
different setups were tested. In the first test the beer after the
fermentation was not treated with enzymes (DDGS blank), within the
second test the beer was treated with the enzyme composition
comprising a xylanase (200 ppm,) and a 1,3-glucanase (200 ppm)
after the fermentation (DDGS treated).
[0171] The trials were performed on a 301 scale. Each test was
carried out as duplicate. The total enzyme concentration added was
400 g/t of beer. After the distillation the fermenter was drained
off and the whole stillage was centrifuged to obtain thin stillage
and the wet cake.
[0172] 301 Corn to Ethanol fermentation with enzyme application and
DDGS production: [0173] pre milled corn <2 mm particle size
[0174] 10 kg corn were mixed with tap water at 35.degree. C. t to
reach a concentration of .about.32% (w/w) [0175] pH range was
5.6-6.0 [0176] temperature was increased to 90.degree. C. [0177] 7
ml alpha-amylase (Liquizyme from Novozymes) were added [0178]
1.Salinity. antifoam was added (30 ml) [0179] incubation for 90 min
at 90.degree. C. and 150 rpm [0180] 12 ml Glucoamylase (Novozymes
Spirizyme Ultra) was added [0181] addition of 300 ppm ((NH4)2SO4)
as nitrogen source [0182] direct inoculation with dry yeast [0183]
pH adjustment to pH 5.5 [0184] fermentation for 62 h at 33.degree.
C. and 150 rpm to obtain the beer [0185] The beer is than treated
with enzymes for 6 hours at 37.degree. C. [0186] after 68 hours
ethanol is removed by distillation obtaining a residue called whole
stillage [0187] afterwards the whole stillage is centrifuged at
3000 rpm resulting in thick stillage and thin stillage [0188]
concentration of the thin stillage to 50% DM by evaporation--The
wet cake is mixed with the thin stillage and dried in a drum dryer
at ca. 120.degree. C. up to a 90% dry matter.
[0189] FIG. 11 shows the amount of thin stillage (supernatant) in %
of whole stillage removed after centrifugation at 3000 rpm for 10
minutes. Here it was shown that a treatment with the enzyme
composition comprising a xylanase and a 1,3-glucanase increases the
dewatering capability by 18.7%.
Example 6
In-Vitro Digestibility of DDGS
[0190] In vitro digestibility assays, (simulating the digestion in
animals) are well accepted to predict the digestibility of feed
stuff.
[0191] Two different setups were tested. In the first test the beer
after the fermentation was not treated with enzymes (DDGS-blank),
for the second test the beer was treated with the enzyme
composition comprising a xylanase (200 ppm) and a 1,3-glucanase
(200 pm) after the fermentation (DDGS treated).
[0192] In the following the In vitro protein digestibility assay is
described:
[0193] DDGS samples are digested by a Pepsin/HCl solution: 0.1 M
HCl with 20 mg/ml Pepsin. (Pepsin from porcine gastric mucosa,
powder, 400-800 units/mg protein, Sigma: P7125) The supernatant is
used for the determination of free amino groups by the TNBS
assay.
Protein Digestion:
[0194] 0.5 g DDGS is mixed with 8 ml of 0.1M HCl with 20 mg/ml
Pepsin. [0195] After mixing with a Vortexer the samples are
incubated for 60 min at 40.degree. C. [0196] After 60 min. the
sample is centrifuged for 20 min at 4000 rpm. [0197] After the
centrifugation the protein is measured in the supernatant Protein
Determination with TNBS-Assay:
[0198] 2,4,6-Trinitrobenzene Sulfonic Acid (TNBSA or TNBS
(2,4,6-Trinitrobenzene Sulfonic Acid) is a rapid and sensitive
assay reagent for the determination of free amino groups. Primary
amines, upon reaction with TNBSA, form a highly chromogenic
derivative, which can be measured at 335 nm (see figure).
Qualitative measurements of amines, sulfhydryls or hydrazides, 3
and quantitative measurements of free-amino groups of L-lysine have
also been obtained using TNBSA.
Preparation of Buffers
TABLE-US-00005 [0199] No Component 1 1% SDS 2 0.1M HCl 3 0.025%
TNBS in 87.5 mM Na-Phosphat pH 8.2 4 100 mM Glycin stock in 1%
SDS
Sampling (in 96-Well-Plate):
[0200] 1. For standard curve: [0201] Glycin start 2500 .mu.M,
2.times. diluted in 1% SDS
TABLE-US-00006 [0201] Glycin row 2500 1250 625 312.5 156.25 78.125
39.0625 0
[0202] 2. Samples, (diluted 1000.times.) with 1% SDS
Reaction (96 Well PCR Plate):
[0202] [0203] 1. 15 .mu.l of Glycin row or samples+90 .mu.l of
buffer No 3 (see buffer list) [0204] 2. PCR plate is incubated in
PCR cycler, program: [0205] 50.degree. C. for 30 min [0206]
4.degree. C. for 10 min [0207] 3. Take out from PCR cycler, mix
properly
Measurement:
[0208] 50 .mu.l of step 3 above are mixed with 50 .mu.l of buffer
No 2 (see buffer list), absorption is measured at 340 nm
[0209] Two different setups were tested. In the first test the beer
after the fermentation was not treated with enzymes (DDGS-blank),
for the second test the beer was treated with an enzyme composition
comprising a xylanase and a 1,3-glucanase as main activities after
the fermentation (DDGS-treated).
[0210] The enzyme treated DDGS was produced with the enzyme
composition comprising a xylanase and a 1,3-glucanase as main
activities with a total concentration of 400 g/t beer. FIG. 12 is
showing the results.
[0211] FIG. 12 shows the release of free amino groups with
different substrates. The DDGS-treated shows an increased
concentration of free amino groups by 20.4%, determined by TNBS
assay
[0212] Therefore it can be clearly shown that compared to the
DDGS-blank the protein digestibility with Pepsin/HCL is increased
by DDGS-treated. This results in an improved nutritional quality of
the DDGS as a by-product of the fermentation process and therefore
is a high quality animal feed.
Example 7
Protein Solubilisation in Water
[0213] Two different setups were tested. In the first test the beer
after the fermentation was not treated with enzymes (DDGS-blank),
for the second test the beer was treated with the enzyme
composition comprising a xylanase (200 ppm) and a 1,3-glucanase
(200 pm) after the fermentation (DDGS treated).
[0214] For that test 0.5 g DDGS was mixed with 8 ml distilled water
and shacked with a Vortexer. The solution was incubated for 60 min
at 40.degree. C. After a centrifugation step (20 min, 4000 rpm,
4.degree. C.) the protein concentration in the supernatant was
analyzed with the TNBS assay (see example 6).
[0215] In FIG. 13 the release of free amino groups of DDGS in water
is shown. The DDGS-treated is showing an increase of free amino
groups by 31.4%. The fiber structure of the DDGS is changed as well
as the yeast cell walls are disrupted resulting in an increased
availability of proteins that can be readily digested in the
animal.
[0216] This result in an improved nutritional quality of the DDGS
as a by-product of a fermentation process and therefore in a high
quality animal feed.
[0217] Some embodiments of the present disclosure pertain to:
[0218] A) Methods to improve the quality of by-products or residues
derived from fermented mash comprising the steps of: i) subjecting
the fermented mash during or after the fermentation to a
composition comprising an enzyme or a mixture of enzymes capable of
degrading one or more fermented mash components, ii) separating the
desired fermentation product, wherein [0219] the fermented mash can
be derived from a process of producing a liquid fermentation
product. [0220] the fermented mash can be derived from a process of
producing a fermentation product utilizing sugar-containing
material as feedstock. [0221] the fermented mash can be derived
from a process of producing a fermentation product utilizing
starch-containing material as feedstock. [0222] the feedstock can
be a cereal. [0223] the feedstock can be selected from the group
consisting of corn, wheat, barley, triticale, cassava, sorghum,
rye, potato, or any combination thereof. [0224] the liquid
fermentation product can be an alcohol, preferably ethanol. [0225]
the composition may comprise an enzyme selected from the group
consisting of amylase, such as alpha-amylase, glucoamylase,
cellulase, beta-glucanase, hemicellulase, such as xylanase,
pectinase, mannanase, and protease, or a mixture thereof. [0226]
the composition may comprise a mannanase. [0227] the composition
may comprises a mannanase and a beta-glucanase. [0228] the desired
fermentation product may be separated by distillation. [0229] B)
Methods for improving the nutritional quality of a by-product or
residue of a fermentative ethanol producing process, comprising the
following steps: [0230] i) Fermentation of fermentable sugars with
a micro-organism [0231] ii) Subjecting the fermented mash to a
composition comprising an enzyme or a mixture of enzymes after the
fermentation process [0232] iii) Separation of the ethanol [0233]
iv) Separation of the by-product or residue, wherein [0234] the
by-product or residue may be selected from the group consisting of
spent brewer's grains, dried distiller's grains, dried distiller's
solubles, distiller's dried grains with solubles, residues of the
cereal processing industry, wheat bran, soybean hulls, citrus pulp,
beet pulp, rice husks or hulls, bagasse, apple pommace, and
mixtures thereof. [0235] step iii) and iv) can be carried out
simultaneously or sequentially. [0236] the composition may comprise
an enzyme selected from the group consisting of amylase, such as
alpha-amylase, glucoamylase, cellulase, beta-glucanase,
hemicellulase, such as xylanase, pectinase, mannanase, and
protease, or a mixture thereof. [0237] the composition may comprise
a mannanase. [0238] the composition may comprise a mannanase and a
beta-glucanase. [0239] C) Methods of dewatering whole stillage
comprising the steps of i) subjecting beer mash to one or more
enzymes capable of degrading one or more beer mash components, ii)
separating ethanol from the beer mash, iii) separating whole
stillage into a solid fraction and a liquid fraction. [0240] D)
Methods of producing ethanol, said method comprising the steps of:
[0241] i) Fermentation of fermentable sugars with a micro-organism
to produce ethanol [0242] ii) Subjecting the fermented mash to a
composition comprising an enzyme or a mixture of enzymes after the
fermentation process [0243] iii) Separation of the ethanol, wherein
[0244] the fermented mash can be subjected to the enzyme
composition after the fermentation step and before the separation
step. [0245] the separation step can be distillation. [0246] the
composition may comprise an enzyme selected from the group
consisting of amylase, such as alpha-amylase, glucoamylase,
cellulase, beta-glucanase, hemicellulase, such as xylanase,
pectinase, mannanase, and protease, or a mixture thereof. [0247]
the composition may comprise a mannanase. [0248] the composition
may comprise a mannanase and a beta-glucanase. [0249] E) Methods to
improve the quality and nutritional composition of by-products or
residues derived from fermentable sugars in an ethanol producing
process comprising the steps of: i) subjecting the fermented mash
to a composition comprising an enzyme or a mixture of enzymes
capable of degrading one or more fermented mash components, ii)
separating the ethanol and the by-products or residues, wherein
[0250] the fermented mash can be subjected to the enzyme
composition after the fermentation step and before the separation
step. [0251] the separation step may be distillation. [0252] the
composition may comprise an enzyme selected from the group
consisting of amylase, such as alpha-amylase, glucoamylase,
cellulase, beta-glucanase, hemicellulase, such as xylanase,
pectinase, mannanase, and protease, or a mixture thereof. [0253]
the composition may comprise a mannanase. [0254] the composition
may comprises a mannanase and a beta-glucanase. [0255] the
fermentation can be performed using a microorganism, such as
bacteria, yeast or fungi. [0256] F) Methods of dewatering whole
stillage comprising one of the above mentioned methods and the
steps of i) subjecting whole stillage to one or more enzymes
capable of degrading one or more whole stillage components, ii)
separating the material into a solid fraction and a liquid
fraction. [0257] G) Use of a hemicellulase for the degradation of
fermented mash components in a fermentative production process.
[0258] H) Use of xylanase, amylase, glucoamylase, cellulase,
hemicellulase, pectinase, or protease, or a mixture thereof, for
the degradation of fermented mash the components in a fermentative
production process. [0259] I) Methods for the manufacturing of an
enzymes composition used for treating fermented mash of in an
fermentative production process to improve the nutritional quality
of a by-product or residue and/or the process ability of the
production process, comprising: [0260] a) inoculating the
by-product or residue with at least one filamentous fungus; [0261]
b) fermenting the by-product or residue; and [0262] c) separating
at least one enzyme from the fermented by-product or residue,
wherein [0263] the filamentous fungus may be selected from the
group consisting of Rhizopus, Aspergillus, Trichoderma, and any
combination thereof. [0264] the by-product or residue can be a
fibrous by-product selected from the group consisting of spent
brewer's grains, dried distiller's grains, dried distiller's
soluble, distiller's dried grains with soluble, wet grains, and
mixtures thereof.
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