Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
CA 02698080 2010-03-26
Title: PROCESS FOR THE EXTRACTION OF MACROMOLECULES FROM A
BIOMASS USING THIN STILLAGE
FIELD OF THE DISCLOSURE
[001] The present disclosure relates to the extraction of
macromolecules
from a biomass using solution comprising thin stillage.
BACKGROUND OF THE DISCLOSURE
[002] Yeast fermentation of starch and sugar containing agriculture
products such as sugar cane, corn, wheat, barley and sugar beet commonly
produces a final solution of ethanol in water and other organic and inorganic
compounds called beer. In the fuel ethanol industry and other distilleries,
the
beer is passed to a distillation column where the ethanol is evaporated
leaving a
complex aqueous solution of materials that includes ions, organic compounds
and other compounds. This material is called stillage. A solid-free clear
solution
arising from stillage is called thin stillage, which comprises a dilute stream
of
organic and inorganic compounds. Due to its high biochemical oxygen demand
(BOD), it is undesirable to dispose of thin stillage without digestion. In
addition,
its relatively low value as a nutrient makes it undesirable to concentrate by
evaporation.
[003] Thin stillage is usually processed by drying to generate solids
called distillers dried grains with solubles (DDGS) that can be used in animal
feeds. To make DDGS, thin stillage has to be concentrated into syrup before
mixing with wet cake. The process to concentrate thin stillage is not energy
efficient as it consumes about 40-45% of the thermal energy required to
evaporate and dry thin stillage, and 30-40% of the electrical energy utilized
in a
dry-grind facility. Accordingly, the energy required to evaporate the large
amount of water entrained in thin stillage is a major cost in the ethanol
industry.
Peyton et al., 2007 (United States Patent 7,267,774) teaches an efficient
process
whereby discharged still bottoms may be filtered in their pasteurized state
under
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sanitary conditions with the water and nutrients directly recovered for
beneficial
human consumption while the solid concentrate is conveyed to a anaerobic
bioreactor that recovers methane to power the pressurized membrane filtration.
The need for this process is driven by the significant BOD of the thin
stillage
making it undesirable to dispose of this stream without digestion and its
relatively
low value as a nutrient making it undesirable to concentrate by evaporation.
[004] Newkirk et a/. (United States Patent 7,090,887) disclose a
multistage extraction protein extraction and recovery process using water and
CaO to adjust pH. Diosady etal. (United States Patent 4,889,921) extracted 100
g of rapeseed meal with 1,800 g of water. Murray (United States Patent
5,844,086) extracted 50 kg of commercial canola meal with 500 L of water. In
all
of these extractions the percent of protein concentrate recovered to water
used in
extraction and processing is less than 3%. Neilsen and Helmer (United States
Patent 5,989,600) describe using phytase to enhance protein recovery after
suspending soy protein concentrate in deionized water at 50 C.
SUMMARY OF THE DISCLOSURE
[005] A process for the extraction of macromolecules from a biomass
using thin stillage, which is a by-product of the ethanol industry, has been
developed. Accordingly, the present disclosure includes a process for the
extraction of macromolecules from a biomass material comprising:
a) contacting the biomass material with a solution comprising thin
stillage to provide a slurry comprising undissolved solids, dissolved
solids and suspended solids; and
b) separating undissolved solids from the slurry to provide a solid
fraction and a liquid fraction; and
C) optionally isolating the dissolved solids from the liquid fraction,
optionally isolating the suspended solids from the liquid fraction and/or
optionally concentrating the liquid fraction,
wherein the macromolecules are comprised in the dissolved solids.
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[006] It is
an embodiment of the application that the thin stillage is
prepared by:
a) fermentation of a carbohydrate-rich biomass material 4ing a
microorganism in an aqueous solution to provide an ethanol-cohtaining
beer;
b) distilling the beer to remove the ethanol to provide a thin stillage;
c) optionally removing suspended solids from the thin stillage; and
d) optionally removing macromolecular solutes from the thin stillage.
[007] In another embodiment, the carbohydrate rich-biomass comprises
starch as a major carbohydrate. In a further embodiment, the carbohydrate rich-
biomass comprises cellulose as a major carbohydrate. In another embodiment,
the carbohydrate rich-biomass comprises sucrose as a major carbohydrate.
[008] In
another embodiment, the carbohydrate rich-biomass is a seed. In
a further embodiment, the carbohydrate rich-biomass is a cereal. In another
embodiment, the cereal is corn, wheat, rice, barley, oats or sorghum or a
mixture
thereof. In a further embodiment, the carbohydrate rich-biomass is a plant
stem
or tuber. In a further embodiment, the stem or tuber is from a potato or sweet
sorghum.
[009] In another embodiment of the disclosure, the starch in the
carbohydrate-rich biomass is converted to glucose by one or more enzymes or
catalysts to produce a fermentable sugar.
[0010] In an
embodiment, the cellulose in the carbohydrate-rich biomass is
depolymerized by one or more enzymes or catalysts to produce a fermentable
sugar.
[0011] In
another embodiment of the disclosure, the microorganism used
in the fermentation of the carbohydrate-rich biomass is a yeast or a
bacterium.
[0012] In a
further embodiment of the disclosure, the suspended solids are
removed from the thin stillage by centrifugation or sedimentation.
[0013] In another embodiment, the macromolecular solutes are removed
from the thin stillage by ultrafiltration or nanofiltration.
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[0014] In an
embodiment of the disclosure, the pH of the thin stillage is
increased above pH 8. In another embodiment, the pH of the thin stillage is
adjusted above pH 8 using waste alkaline solution from biodiesel production.
In
a further embodiment, the waste alkaline solution from biodiesel production is
a
glycerol/potassium hydroxide solution.
[0015] In
another embodiment of the disclosure, the pH of the thin stillage
is decreased below pH 5.
[0016] In a
further embodiment of the disclosure, the thin stillage further
comprises an additive. In another embodiment, the additive is a salt,
detergent or
zwitterions or a mixture thereof.
[0017] In
another embodiment, the slurry of the biomass material and thin
stillage is heated to improve extraction of the macromolecules from the
biomass.
[0018] In
another embodiment of the disclosure, the undissolved solids in
the slurry of the biomass material and thin stillage are separated by
filtration or
centrifugation.
[0019] In a
further embodiment, the dissolved solids in the slurry of the
biomass material and thin stillage are isolated by ultrafiltration,
diafiltration,
reverse osmosis or nanofiltration. In another embodiment, the dissolved solids
in
slurry of the biomass material and thin stillage are isolated by evaporation.
In an
embodiment, the dissolved solids are isolated by precipitation.
[0020] In a
further embodiment of the disclosure, the dissolved solids in
slurry of the biomass material and thin stillage are precipitated by a change
in pH
or ionic strength.
[0021] In
another embodiment, the contacting of the biomass material with
the thin stillage is conducted in a continuous process. In another embodiment,
the contacting of the biomass material with the thin stillage is conducted in
a
batch process.
[0022] In
another embodiment, the contacting of the biomass material with
the thin stillage is conducted in a countercurrent process. In
another
embodiment, the contacting of the biomass material with the thin stillage is
combined with the preparation of the thin stillage in a continuous process.
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[0023] In another embodiment, the present disclosure also includes a
macromolecular concentrate or isolate produced in accordance with the process
of the disclosure. In another embodiment, the macromolecules comprise
proteins, optionally a protein isolate or a protein concentrate.
[0024] Other features and advantages of the present invention will become
apparent from the following detailed description. It should be understood,
however, that the detailed description and the specific examples while
indicating
preferred embodiments of the invention are given by way of illustration only,
since various changes and modifications within the spirit and scope of the
invention will become apparent to those skilled in the art from this detailed
description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] The application will now be described in greater detail with
reference to the following drawings in which:
[0026] Figure 1 is a graph showing mucilage extraction using NaHCO3
with respect to the time of the extraction.
DETAILED DESCRIPTION
(I) DEFINITIONS
[0027] The term "biomass material" as used herein refers to any
biomass
in which desirable macromolecules, such as proteins, can be extracted.
Examples of biomass include, but are not limited to, an oilseed, plant matter,
animal matter, etc. Examples of oilseeds include, but are not limited to,
canola
seed, rapeseed, mustard seed, flax seed, soybean, nuts such as peanuts, hemp
seed, linseed, cotton seed, etc. In an embodiment, the biomass material is a
ground, defatted oilseed meal.
[0028] The term "thin stillage" as used herein refers to a complex
aqueous
solution including, but not limited to, ions, organic compounds and other
compounds, which is often obtained from the fuel ethanol industry as a common
waste byproduct. Thin stillage is produced from the fermentation, by yeast
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and/or other microorganisms, of starches and sugar, which results in an
ethanol
containing slurry called a beer. After distillation of the beer to remove the
ethanol, a slurry called stillage remains. After filtering the solids from the
stillage,
a thin stillage is obtained, which is a complex aqueous solution. Compounds
that
have been identified in a thin stillage include, but are not limited to,
carbohydrates, fiber, protein, ash, amino acids, lipids, yeast, yeast
metabolites,
bacteria, bacterial metabolites, fungi, wheat metabolites and minerals. Yeast
metabolites include, but are not limited to, compounds selected from glycerol,
ethanol, succinic acid, glycerophosphorylcholine and phenylethyl alcohol.
Bacterial metabolites include, but are not limited to, compounds selected from
isopropanol, acetic acid, lactic acid and 1,3-propanediol. Wheat metabolites
include, but are not limited to, betaines. Minerals include, but are not
limited to,
calcium chloride, sodium chloride, potassium sulphate, sodium nitrate,
magnesium hydroxide, sodium sulphate and potassium hydroxide. In another
embodiment, synthetic thin stillages are also produced by combining many, or
all,
of the above identified compounds.
[0029] The term "macromolecule" as used herein refers to any compound
present in the biomass which is capable of being extracted with thin stillage.
In
an embodiment, the macromolecules comprise protein, peptides, gums,
mucilaginous compounds, polyphenolic compounds and/or complex polymers of
carbohydrates and gums. In another embodiment, the macromolecules comprise
protein and/or peptides.
[0030] The term "contacting" as used herein refers to the manner in
which
the solution comprising the thin stillage and the biomass material are
intimately
combined so that the solution extracts macromolecules, such as proteins, from
the biomass. For example, the solution comprising the thin stillage is stirred
with
the biomass to ensure intimate contact of the solution with the macromolecules
in
the biomass, and results in a slurry comprising undissolved solids, dissolved
solids and suspended solids.
[0031] The term "slurry" as used herein refers to a mixture of thin
stillage
and the biomass material containing macromolecules, that has been sufficiently
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mixed to form a mixture comprising undissolved solids, dissolved solids and
suspended solids.
[0032] The term "undissolved solids" as used herein refers to any
compounds in the biomass material which are substantially not dissolved by the
thin stillage and typically settle to the bottom of the reaction vessel upon
standing.
[0033] The term "dissolved solids" as used herein refers to any
compounds in the biomass material which are substantially dissolved by the
thin
stillage.
[0034] The term "suspended solids" as used herein refers to any
compounds in the biomass material which do not dissolve in the thin stillage,
but
have a molecular weight such that they substantially form a suspension in the
thin stillage.
[0035] The terms "separating", "removing" and "isolating" as used
herein
refer to any method of separating and isolating one type of material from
another,
typically the separation of a solid material from a liquid material or the
separation
of one or more solid or liquid materials from another. The selection of
methods
to be used for the separation of solid materials from liquid materials will
depend
on the size of the solid particles to be separated, as would be known to a
person
skilled in the art, and include, for example filtration, nanofiltration,
osmosis,
centrifugation, sedimentation, precipitation, etc. The selection of methods to
be
used for the separation of one or more liquids or solids from each other will
depend on the physical characteristics of the materials, as would be known to
a
person skilled in the art, and include all forms of chromatography,
precipitation,
recrysta II ization, etc.
[0036] The term "solid fraction" as used herein refers to the combined
undissolved solids that have been separated from the slurry.
[0037] The term "liquid fraction" as used herein refers to the
solution of
thin stillage containing both the dissolved solids and suspended solids.
[0038] The phrase "alkaline solution from biodiesel production" as used
herein refers to a glycerol by-product of the bio-diesel industry. It will be
7
=
understood to those skilled in the art that macromolecule extractions, such as
protein extractions, using aqueous solutions are improved when the extraction
is
performed at an alkaline pH. Biodiesel is a fuel produced from triglyceride
oils
that can be used to fuel diesel engines. Biodiesel is commonly defined as the
monoester of a lower aliphatic alcohol and a fatty acid. It is typically
produced by
trans-esterification (alcoholysis) of triglyceride molecules using a catalyst
and a
monohydric alcohol (methanol, ethanol, etc.) to form monoesters and glycerol.
The trans-esterification reaction utilizes three moles of alcohol to react
with one
mole of triglyceride. The reaction yields three moles of fatty acid ester and
one
mole of glycerol. Biodiesel production commonly utilizes either hydroxide or
methoxide as the catalyst. Sodium or potassium hydroxide is dissolved in
methanol and forms methoxide ions, the actual catalytic agents. Accordingly, a
by-product of the bio-diesel industry is alkaline glycerol, which is therefore
utilized in an embodiment of the present disclosure to adjust the pH of the
protein
extraction process. Such alkaline solutions are described in W02009/067809 to
Reaney et al.
[0039] Terms of degree such as "substantially", "about" and
"approximately" as used herein mean a reasonable amount of deviation of the
modified term such that the end result is not significantly changed. These
terms
of degree should be construed as including a deviation of at least 10% of the
modified term if this deviation would not negate the meaning of the word it
modifies.
(II) PROCESS OF THE DISCLOSURE
[0040] The ethanol fuel industry produces thin stillage as a by-
product of
the fermentation of starches and carbohydrates. Thin stillage is a complex
aqueous solution including, but not limited to, ions, organic compounds and
other
compounds. The process of concentrating thin stillage by evaporation of the
water takes large amounts of energy. During the ethanol production process,
the
temperature of the thin stillage rises to about 80-85 C as a result of the
ethanol
distillation process. Accordingly, in an embodiment, as protein extractions
are
optionally conducted at increased temperatures, there are large energy savings
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when thin stillage is used as a macromolecule (such as a protein) extraction
medium. In addition, in an embodiment, there are large water savings when thin
stillage is used as the extraction medium, for example, in protein extraction,
as
this industry uses large amounts of water.
[0041] Furthermore, in an
embodiment, thin stillage contains
minerals [Ojowi et al., Canadian Journal of Animal Science, 1996, Vo, 76, 547-
553; Mustafa et al., Animal Feed Science and Technology, 1999, 80, 247-256]
which may enhance protein extraction, such as the proteins napin and
cruciferin.
It also contains yeast cells, soluble nutrients and grain protein molecules
(Wheals
et al., Trend in Biotechnology, 1999, 17(12), 482-487) as protein sources.
Therefore, in an embodiment, the protein content of the extracted
macromolecules, may increase. Moreover, ethanol residue remains in the thin
stillage after the distillation process (Wilkie etal., Biomass and Bioenergy,
2000,
19, 63-102), along with other organic compounds (Dowd et al., Journal of
Agriculture and Food Chemistry, 1994, 42, 283-288; Wilkie et al., Biomass and
Bioenergy, 2000, 19, 63-102), which might have an inhibitory effect on
myrosinase activity (Botti et al., The Journal of Biochemical Chemistry, 1995,
270(35), 20530-20535) in defatted meal, which in an embodiment, would allow
more efficient isolation of intact glucosinolates.
[0042] In an
embodiment, a process for the extraction of macromolecules
from a biomass using thin stillage, which is a by-product of the ethanol
industry
has been developed. Accordingly, the present disclosure includes a process for
the extraction of macromolecules from a biomass material comprising:
a) contacting the biomass material with a solution comprising thin
stillage to provide a slurry comprising undissolved solids, dissolved
solids and suspended solids; and
b) separating undissolved solids from the slurry to provide a solid
fraction and a liquid fraction; and
c) optionally isolating the dissolved solids from the liquid fraction,
optionally isolating the suspended solids from the liquid fraction and/or
optionally concentrating the liquid fraction,
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wherein the macromolecules are comprised in the dissolved solids.
[0043] It is
an embodiment of the application that the thin stillage is
prepared by:
a) fermentation of a carbohydrate-rich biomass material using a
microorganism in an aqueous solution to provide an ethanol-containing
beer;
b) distilling the beer to remove the ethanol to provide a thin stillage;
c) optionally removing suspended solids from the thin stillage; and
d) optionally removing macromolecular solutes from the thin stillage.
[0044] In another embodiment, the carbohydrate rich-biomass comprises
starch as a major carbohydrate. In a further embodiment, the carbohydrate rich-
biomass comprises cellulose as a major carbohydrate.
[0045] In
another embodiment, the carbohydrate rich-biomass comprises
sucrose as a major carbohydrate.
[0046] In another embodiment, the carbohydrate rich-biomass is a seed. In
a further embodiment, the carbohydrate rich-biomass is a cereal. In another
embodiment, the cereal is corn, wheat, rice, barley, oats or sorghum or a
mixture
thereof. In a further embodiment, the carbohydrate rich-biomass is a plant
stem
or tuber. In a further embodiment, the stem or tuber is from a potato or sweet
sorghum.
[0047] In
another embodiment of the disclosure, the starch in the
carbohydrate-rich biomass is converted to glucose by one or more enzymes or
catalysts to produce a fermentable sugar.
[0048] In an
embodiment, the cellulose in the carbohydrate-rich biomass is
depolymerized by one or more enzymes or catalysts to produce a fermentable
sugar.
[0049] In
another embodiment of the disclosure, the fermentation
microorganism is a yeast or a bacterium.
[0050] In a
further embodiment of the disclosure, the suspended solids are
removed from the thin stillage by centrifugation or sedimentation.
CA 02698080 2010-03-26
[0051] In another embodiment, the macromolecular solutes are removed
from the thin stillage by ultrafiltration or nanofiltration.
[0052] In another embodiment of the disclosure, the pH of the thin
stillage
is adjusted to increase the efficiency of the extraction of macromolecules
from
the biomass material. It will be understood by those skilled in the art that
by
adjusting the pH of the thin stillage, for example to a pH below 5 or a pH
above 8,
a higher concentration of macromolecules, such as protein, are extracted from
the biomass material due to the increased solubility of the macromolecules.
[0053] In an embodiment of the disclosure, the pH of the thin
stillage is
increased above pH 8. In another embodiment, the pH of the thin stillage is
adjusted above pH 8 using waste alkaline solution from biodiesel production.
In
a further embodiment, the waste alkaline solution from biodiesel production is
a
glycerol/potassium hydroxide solution.
[0054] In another embodiment of the disclosure, the pH of the thin
stillage
is decreased below pH 5.
[0055] In a further embodiment of the disclosure, the thin stillage
further
comprises an additive. In another embodiment, the additive is a salt,
detergent or
zwitterions or a mixture thereof. It will be understood by those skilled in
the art
that the addition of additives, such as salt (for example, sodium chloride)
increases the efficiency of the extraction of the macromolecules from the
biomass material into the thin stillage by increasing the solubility of the
macromolecules, such as protein, in the thin stillage.
[0056] In another embodiment, the slurry formed by the biomass
material
and the thin stillage is heated. In another embodiment, the slurry is heated
to
temperature of between about 10 C to about 80 C, optionally between about
20 C to about 60 C, optionally between about 40 C to about 60 C, optionally
between about 40 C to about 44 C.
[0057] In another embodiment of the disclosure, the undissolved
solids are
separated from the slurry by filtration or centrifugation.
[0058] In a further embodiment, the dissolved solids are separated and
isolated from the slurry by ultrafiltration, diafiltration, reverse osmosis or
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nanofiltration. In another embodiment, the dissolved solids are isolated by
evaporation. In an embodiment, the dissolved solids are isolated by
precipitation.
[0059] In a
further embodiment of the disclosure, the dissolved solids are
precipitated by a change in pH or ionic strength.
[0060] In another
embodiment, the contacting of the biomass material with
the thin stillage is conducted in a continuous process. In another embodiment,
the contacting of the biomass material with the thin stillage is conducted in
a
batch process.
[0061] In
another embodiment, the contacting of the biomass material with
the thin stillage is conducted in a countercurrent process. In
another
embodiment, the contacting of the biomass material with the thin stillage is
combined with the preparation of the thin stillage in a continuous process.
[0062] In
another embodiment, the present disclosure also includes a
macromolecular concentrate produced in accordance with the process of the
disclosure. In another embodiment, the macromolecules comprise proteins.
[0063] In
another embodiment, the biomass material comprises an
oilseed. In another embodiment, the oilseed comprises a defatted oilseed meal.
In a further embodiment, the defatted oilseed meal comprises defatted canola
seed meal, defatted rapeseed meal, defatted mustard seed meal, defatted flax
seed meal, defatted soybean meal, defatted peanut meal, defatted hemp seed
meal, defatted linseed meal or defatted cotton seed meal.
[0064] In
another embodiment, the macromolecules comprise protein,
peptides, gums, mucilaginous compounds, polyphenolic compounds and
complex polymers of carbohydrates and gums. In another embodiment, the
macromolecules comprise protein and/or peptides.
[0065] In
another embodiment of the disclosure, the thin stillage comprises
compounds selected from carbohydrates, fiber, protein, amino acids, lipids,
yeast, yeast metabolites, bacteria, bacterial metabolites, fungi, wheat
metabolites
and minerals. In
another embodiment, the yeast metabolites comprise
compounds selected from glycerol, ethanol, succinic acid,
glycerophosphorylcholine and phenylethyl alcohol. In a further embodiment, the
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bacterial metabolites comprise compounds selected from isopropanol, acetic
acid, lactic acid and 1,3-propanediol. In
another embodiment, the wheat
metabolites comprise betaine.
[0066] In
another embodiment, before using as the extraction medium for
the macromolecules, the thin stillage is filtered to remove other
macromolecules
which remain after the fermentation process which generates the thin stillage.
[0067]
Certain embodiments of the invention are disclosed below by way
of example.
EXAMPLES
[0068] Mustard seed (Brassica juncea (L.) Czem) was obtained from
Agriculture aand Agri-Food Canada, Saskatoon Research Centre, Saskatoon,
SK. All seed was from the 2006 harvest and was grown on plots near
Saskatoon. Thin stillage (wheat basis) was provided by Pound-Maker Agventures
Ltd., Lanigan, SK. Samples of thin stillage were taken on four collection
dates
(May 18, May 27, May 28 and June 1, 2007) and were stored at 4 C (for up to 4
months) until used. However, micro-organisms can possibly grow over the 4
month storage period. Prior to all analysis of physical properties, chemical
properties, chemical constituents, micro-organism content and ion content,
samples were centrifuged at 1,053 x g for 20 minutes at 4 C (Model Avanti
J-E, Beckman Coulter Canada Inc., Mississauga, ON). Glycerol containing
approximately 10 per cent KOH was provided from an industrial biodiesel
processor (Milligan Biotechnology Inc., Foam Lake, SK).
Example 1: Defatted Meal Preparation
[0069] Mustard seed was extracted mechanically using a continuous
screw expeller (Komet, Type CA59 C; IBG Monforts Oekotec GmbH & Co.,
Monchengladbach, Germany) operated at a speed of 6.5 (approximately 93 rpm)
using a 6-mm choke. Oil remaining in the presscake was removed using
hexane as a solvent (Milanova et al., 2006; Oomah et al., 2006), and the
residual hexane in the defatted meal was removed in a fume hood overnight.
Defatted meal was analyzed for protein and oil content.
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[0070] Protein content was determined on 0.5 g samples using the
Kjeldahl method [modified from method 981.10 of the A.O.A.0 (1990)]. Samples
were digested by heating with concentrated H2SO4 in a heating/digestion block
using a package of Kjeldahl digestion mixture #200 as a catalyst. After
digestion,
samples were distilled using a steam distillation unit (Model 320, BOchi
Analytical
Inc., New Castle, DE) with 30% (w/v) NaOH. Boric acid (4%) was used to trap
ammonia from the distillation. The distillate was titrated with 0.2N HCI using
N-
Point indicator as an indicator. The nitrogen content was calculated using the
equation:
% N = (mL of 0.2N HCI sample ¨ mL HCI blank) x normality of HCI x 0.014 x
(1/sample weight x 100).
The % N of the sample was converted to % protein content by multiplying %N by
6.25.
[0071] Oil content was determined using a Goldfisch Extractor (Model
22166B, LabConCo Corporation, Kansas City, MO) [modified from method
960.39(a) of the A.O.A.0 (1990)]. Approximately 20 g of sample was ground
using a coffee grinder to pass through a 1.0-mm screen (approximately 30
seconds). Three grams of ground sample was weighed on a filter paper
(Whatman No. 4) and folded. The samples were placed in cellulose thimbles (25
X 80 mm, Ahlstrom AT, Holly Spring, PA). Samples were extracted for 6 hours
using 50 mL of hexane as solvent. The hexane was distilled from the oil
extraction beakers, after which the beakers were heated at low temperature (30-
40 C) using a hot plate placed in a fume hood. The beakers were then
transferred to an oven (105 C) for 30 minutes and then allowed to cool to room
temperature (approximately 25 C) in a desiccator.
[0072] Moisture content was determined by heating a weighed sample [1
g
of ground sample using a coffee grinder to pass through 1.0-mm screen
(approximately 30 seconds)] at 100-102 C for 16-18 hours or until the weight
of
the sample was constant [modified from method 950.46 B.a, of the A.O.A.0
(1990)]. The samples were allowed to cool to room temperature in a desiccator
for at least 1 hour before weighing.
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Example 2: Extraction of mustard (Brassica juncea) protein with water
(comparative example)
[0073] Five
grams of ground defatted B. juncea meal was mixed with 150
mL of water adjusted salt concentration to 1M. The pH of the mixture was
adjusted to pH 10 using KOH dissolved in glycerin (a co-product of biodiesel
production). The solution was then stirred for two hours. Subsequently, the
solution was centrifuged at 5,000 x g for 10 minutes and supernatant was taken
for dialysis using Spectra/Por molecular porous membrane tubing at 3,500
molecular weight cut off (MWCO) in the ratio 1:1000 of supernatant to
distilled
water. Water exchange with fresh deionized water was repeated 3 times a day,
until the conductivity of permeate water was equal to that of deionized
distilled
water after 8 hours of dialysis (approximately 5 days). The solution after
dialysis
was freeze dried. The extracted protein had protein content (Nx6.25)
approximately 105% by weight with the protein exaction efficiency being
approximately 60%. After that extracted protein was used to examine SDS-
PAGE, peptide sequencing, amino acid composition, in vitro digestibility, and
lysine availability. The results form SDS-PAGE showed that the molecular
weight
of extracted protein from water was approximately 14, 18-20, 20-22, 34, and 55
kDa. In addition, the results of peptide sequencing, amino acid composition,
and
in vitro digestibility and lysine availability are presented in table 1, 2,
and 3
respectively.
Example 3: The extraction of mustard (Brassica juncea) protein with thin
stillage
[0074] Five
grams of ground defatted B.juncea meal was mixed with 150
mL of centrifuged filtered thin stillage (centrifuge at 1,053 x g for 20
minutes at
4 C, filter through 3,000 molecular weight cut off membrane), which salt level
was adjusted to 1M. The pH of the mixture was adjusted to pH 10 using KOH
dissolved in glycerin (a co-product of biodiesel production). After that the
solution
CA 02698080 2010-03-26
was stirred for two hours. Subsequently, supernatant was taken for dialysis
using
Spectra/Por molecular porous membrane tubing 3,500 MWCO in the ratio 1:1000
of supernatant to distilled water. Water exchange with fresh deionized water
was
repeated 3 times a day, until the conductivity of permeate water was equal to
that
of deionized distilled water after 8 hours of dialysis (approximately 5 days).
The
protein solution after dialysis was freeze dried. The extracted protein has
protein
content (Nx6.25) approximately 96% by weight with the protein exaction
efficiency approximately 56%. Extracted protein was taken for examining gel
electrophoresis, animo acid composition, in-vitro digestibility, and lysine
availability. The results from SDS-PAGE showed that protein extracted from
thin
stillage had molecular weight approximately, 14, 18-20, 20-22, 34, and 55
kDa.,
amino acid content, in-vitro digestibility, and lysine availability of
extracted protein
are reported in table 4 and 5.
Example 4: The extraction of Flax mucilage with thin stillage.
[0075] 5 grams of flax seeds were mixed with 40 mL of 0.5M sodium
bicarbonate (NaHCO3). The extraction time was varied from 15, 30, 45, and 60
minutes. The mucilage extraction was done at 50 C and 3 times extraction
countercurrently. Each time of extraction the viscous liquid was separated
from
the flax seeds using syringes and the rest of viscous liquid was separated
using
a centrifuge at rpm for 40 minutes at 25 C. The liquid from 3 times extraction
was
collected and the centrifuged thin stillage (3000 rpm for 20 minutes at 4 C)
was
added into the viscous liquid at 1:1 ratio. The viscosity of the liquid was
measured at 25 C using a Shell cup No.1. After that 95% ethanol was added to
the liquid at the ratio 1:1. The solution then was stirred for 1 hour for the
first
washing. The mucilage was separated from the solution using a centrifuge at
6000 rpm for 10 minutes at 4 C. The second wash of mucilage was done using
approximately 110 mL of 95% ethanol stirring for 30 minutes. The mucilage was
separated from ethanol using a centrifuge at 6000 rpm for 10 minutes at 4 C.
The mucilage was then transferred to the moisture tint and was placed in the
16
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over at 105 C overnight was weigh the dry weight. Figure 1 shows a graph of
the grams of mucilage extraction versus the length of the extraction.
[0076] 5 grams of flax seeds were mixed with centrifuged thin stillage
(pH
of thin stillage was adjusted to 7 using 30%(w/w) NaOH and centrifuged at 4000
rpm for 40 minutes at 4 C). 0.5M of NaHCO3 was added into centrifuged thin
stillage). The mucilage was extracted for 30 minutes at 50 C 3 times
extraction
countercurrently. Each time of extraction the viscous liquid was separated
from
the flax seed using syringes and the rest of viscous liquid was separated
using a
centrifuge at rpm for 40 minutes at 25 C. The viscous liquid 3 extractions was
collected. The distilled water was added into the viscous liquid at 1:1 ratio.
The
viscosity of the liquid was measured at 25 C using a Shell cup No.1. After
that
95% ethanol was added into the liquid at the ratio 1:1 and the solution was
stirred
for 1 hour. The mucilage was separated from the solution using a centrifuge at
6000 rpm for 10 minutes at 4 C. The second wash of mucilage was done using
approximately 110 mL of 95% ethanol stirring for 30 minutes. The mucilage was
separated from ethanol using a centrifuge at 6000 rpm for 10 minutes at 4 C.
The mucilage was then transferred to the moisture tint and was placed in the
over at 105 C overnight was weigh the dry weight. The results of mucilage
extraction using water or thin stillage are present in Table 6.
Example 5: Extraction of Oat beta glucan with thin stillage
[0077] Oat groats (150 g) were abraded until approximately 10% of
groat
weight had been removed with a laboratory Satake mill, fitted with a #30 stone
and a 1.00 mm slotted screen (approximately 20 second). Abraded oat groats
(40 g) were steeped in 100 mL of 0.1% sodium metabisulphite at 50 C for 4
hours during which time the groats rapidly imbibed water to the extent of
their
own weight. Steeped abraded oat groats were ground with 50% ethanol 3 times
(2 minutes/time) using a blender. After each grinding step the mixture was
passed through a 250 micron screen to separate the course bran from the finer
flour fraction. Subsequently the bran was washed twice with 50% ethanol (at a
ratio of 1 part bran to 5 parts solution; 20 minutes/wash) the bran was
recovered
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CA 02698080 2010-03-26
by screening after each wash. The bran was air dried to yield an oat bran
which
contains 14-20% of beta glucan.
[0078] Oat bran (1.25 g) was slurried in 120 mL of distilled water at
80-
90 C for 1 hour to re-hydrated the gum. The slurry was filtered using grade 50
Veratec Graphic Art cheese cloth. The viscosity of the gum was measured at
25 C using a Shell cup No.1. The gum was mixed with 95% ethanol and stirred
for 20 minutes. After that the precipitated gum was separated from solution by
centrifugation (7,000 rpm for 40 minutes at 4 C). The precipitated gum was
washed 2 more times then was transferred to a foil container for drying. The
gum
was dried overnight at 105 C.
[0079] Oat bran (1.25g) was slurried in 120 mL of centrifuged thin
stillage
(pH of the thin stillage was adjusted to 7 using 30% (w/w) NaOH and
centrifuged
at 4000 rpm for 40 minutes at 4 C) at 80-90 C for 1 hour to re-hydrated the
gum.
The slurry was filtered using cheese cloth. The viscosity of the gum was
measured at 25 C using a Shell cup No. 1. The gum was mixed with 95%
ethanol and stirred for 20 minutes. After that the precipitated gum was
separated
from solution using a centrifuge at 7,000 rpm for 40 minutes at 4 C. The
precipitated gum was washed 2 more times then was transferred to foil
container
for drying. The gum was dried overnight at 105 C. The results of beta glucan
extraction using water and thin stillage are shown in Table 7. The viscosity
of the
gum recovered by thin stillage extraction was approximately the same as the
product recovered from water extraction.
Example 6: Extraction and Recovery of Small Particle Starch Using Thin
Stillage
[0080] Dehulled buckwheat (25 g) was steeped in 50 mL of 1% (w/w)
sodium metabisulphite at 45 C for 72 hours. The steeped buckwheat was then
ground three times in a blender using 75 mL of distilled water (2
minutes/time).
The slurry was passed through 40, 60, and 400 mesh screens respectively. The
starch suspension was then centrifuged at 7,000 rpm for 40 minutes at 4 C. The
solution was drained and the starch pellet produced by centrifugation was
washed twice with distilled water adjusted pH to 9.5 (using 0.1M NaOH) in a
ratio
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1 part solid to 3 parts washing solution (20 minutes/wash). After each wash,
the
starch solution was centrifuged (7,000 rpm; 40 minutes; 4 C). Washed starch
was mixed with distilled water and the pH was adjusted to 9.5 (ratio 1:3) at
40 C
for 2 hours (checked pH every 20 minutes and pH was adjusted using 0.1M
NaOH). Starch solution was settled at 4 C for 90 minutes. After that the
supernatant was drained. Starch was washed by mixing with distilled water
(ratio
1:3) 20 minutes and settling in the fridge for 90 minutes and the supernatant
was
drained. Starch was then rewashed 2 more times and was dried out using air
dry.
The starch was weighted, analyzed for protein content and particle size.
[0081] Buckwheat (25 g) was steeped in 50 mL of 1% (w/w) sodium
metabisulphite at 45 C for 72 hours. The steeped buckwheat was ground with
thin stillage that had been neutralized to pH 7 then centrifuged (4000 rpm for
40
minutes at 4 C) to remove suspended solids. The steeped buckwheat was then
ground three times in a blender using 75 mL of stillage (2 minutes/time). The
starch and bran slurry was passed through 40, 60, and 400 mesh screens in
sequence. The starch starch slurry (material that passed the 400 mesh screen)
was then centrifuged at 7,000 rpm for 40 minutes at 4 C. After centrifugation
starch pellet was washed with thin stillage adjusted pH to 9.5 at a ratio 1
part
starch pellet to three parts thin stillage 2 times (20 mintues/time). After
each
wash, the starch suspension was centrifuged at 7,000 rpm for 40 minutes at 4
C.
Washed starch was mixed with centrifuged thin stillage adjusted pH to 9.5
(ratio
1:3) at 40 C for 2 hours (checked pH every 20 minutes). Starch solution was
settled in a fridge for 90 minutes. After that the supernatant was drained
out.
Starch was washed by mixing with centrifuged thin stillage adjusted pH to 7
(ratio
1:3) 20 minutes and settling in the fridge for 90 minutes and the supernatant
was
drained. Starch was then rewashed 2 more times (first time with centrifuged
thin
stillage and second time with distilled water). The starch was air dried and
then
weighed. The protein content of the starch was estimated from the nitrogen
content and particle size was determined by light scattering. The results of
starch
extraction using water or thin stillage are presented in Table 8.
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TABLE 1: Peptide Sequencing of Protein Extracted with Water
Subunit Actual
Calculated Sequence
mass Fragment sequence mass
position
mass (Da) assignment
(kDa) (Da)
EFQQAQQHLR 1155.5785 1156.6826 Allergen 12-20
(SEQ ID NO: 1)
B.juncea 1-
E
IYQTATHLPR
(SEQ ID NO: 2) 1198.6458 1199.7471 Allergen 100-
109
14
IEVWDHHAPQLR B.juncea 1-
(SEQ ID NO: 3)
1499.7633 1500.8756 Cruciferin 50-61
GLPLEVISNGYQISPQEAR
2070.0745 2071.2039 Cruciferin 420-438
(SEQ ID NO: 4)
18-20 GLPLEVISNGYQISPQEAR 2070.0745 2071.2136 Cruciferin 338-386
(SEQ ID NO: 5)
GLPLEVISNGYQISLEEAR
(SEQ ID NO: 6) 2087.0898 2088.1885 Cruciferin 66-84
20-22
2070.0745 2071.1917 Cruciferin 368-386
GLPLEVISNGYQISPQEAR
(SEQ ID NO: 7)
CSGFAFER
(SEQ ID NO: 8)
972.4124 973.4891 Cruciferin 62-69
34 VQGQFGVIRPPLR 1465.8518 1466.9407 Cruciferin 251-263
(SEQ ID NO: 9)
1499.7633 1500.8281 Cruciferin 50-61
IEVWDHHAPQLR
(SEQ ID NO: 10)
GPFQVVRPPLR
(SEQ ID NO: 11)
1264.7404 1265.7958 Cruciferin 288-298
VQGQFGVIRPPLR
(SEQ ID NO: 12) 1465.8518 1466.9131 Cruciferin 251-
263
1499.7633 1500.8154 Cruciferin 50-61
IEVWDHHAPQLR
(SEQ ID NO: 13) 2070.0745 2071.1272 Cruciferin 420-
438
GLPLEVISNGYQISPQEAR
(SEQ ID NO: 14)
5
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TABLE 2: Amino Acids of Protein Extracted with Water
Protein extracted
Amino acid
from water
Cysteine 5.19 0.2
Asparagine 5.99 0.1
Methionine 2.26 0.03
Threonine 3.45 0.2
Serine 4.24 0.04
Glutamic acid 23.04 0.7
Glycine 4.94 0.09
Alanine 4.32 0.08
Valine 3.41 2
lsoleucine 3.81 0.06
Leucine 7.45 0.07
Phenylalanine 4.07 0.7
Histidine 4.54 0.08
Lysine 5.90 0.3
Arginine 7.56 0.1
Tryptophan
Tyrosine
Aspartic acid
Praline
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TABLE 3: In vitro Digestibility and Lysine Availability of Protein Extracted
with Water
In vitro digestibility Lysine availability
Extracted protein
74.53 0.5 41.84 4
from water
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TABLE 4: Amino Acids of Protein Extracted with Thin Stillage
Protein extracted
Amino acid
from thin stillage
Cysteine 5.27 0.01
Asparagine 5.41 0.2
Methionine 2.18 0.2
Threonine 3.13 0.05
Serine 3.91 0.1
Glutamic acid 22.23 0.1
Glycine 4.59 0.07
Alanine 3.94 0.08
Valine 5.97 0.2
lsoleucine 3.43 0.01
Leucine 6.62 0.05
Phenylalanine 3.64 0.08
Histidine 4.52 0.05
Lysine 5.18 0.02
Arginine 7.02 0.05
Tryptophan
Tyrosine
Aspartic acid
Proline
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TABLE 5: In vitro Digestibility and Lysine Availability of Protein Extracted
with Thin Stillage
In vitro digestibility Lysine availability
Extracted protein
74.89 0.8 43.01 0.3
from water
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TABLE 6: Mucilage Extraction Using Water or Thin Stillage
Viscosity of the
Grams of mucilage
Solvent Time (minute) mucilage
(dry weight)
(centipoise)
15 0.61 0.03 3.15 0.07
30 0.66 0.01 3.20 0.00
0.5M NaHCO3
45 0.67 0.01 3.30 0.00
60 0.68 0.01 3.35 0.07
Thin stillage 30 0.58 0.01 3.25 0.07
CA 02698080 2010-03-26
TABLE 7: Oat Groat Extraction Using Water or Thin Stillage
Grams of gum Viscosity of the
gum
Solvent
(dry weight) (centipoise)
Water 0.31 0.00 8.85 0.49
Thin stillage 0.54 0.01 8.50 0.00
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TABLE 8: Extraction of Buckwheat Using Water or Thin Stillage
Starch content Protein content Particle
size
Solvent
(Y0w/w) (70w/w) (pm)
Water 58.82 4.53 1.17 0.08 15.53
1.48
Thin stillage 51.11 1.90 2.31 0.10 19.97
3.36
27
