CA2698080C - Process for the extraction of macromolecules from a biomass using thin stillage - Google Patents
Process for the extraction of macromolecules from a biomass using thin stillage Download PDFInfo
- Publication number
- CA2698080C CA2698080C CA2698080A CA2698080A CA2698080C CA 2698080 C CA2698080 C CA 2698080C CA 2698080 A CA2698080 A CA 2698080A CA 2698080 A CA2698080 A CA 2698080A CA 2698080 C CA2698080 C CA 2698080C
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- Canada
- Prior art keywords
- thin stillage
- biomass
- solids
- carbohydrate
- biomass material
- Prior art date
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- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- 101710202365 Napin Proteins 0.000 description 1
- 241001632427 Radiola Species 0.000 description 1
- 240000000111 Saccharum officinarum Species 0.000 description 1
- 235000007201 Saccharum officinarum Nutrition 0.000 description 1
- 235000019764 Soybean Meal Nutrition 0.000 description 1
- 108010073771 Soybean Proteins Proteins 0.000 description 1
- 235000021536 Sugar beet Nutrition 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000006136 alcoholysis reaction Methods 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000000149 argon plasma sintering Methods 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 229960003237 betaine Drugs 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- 239000004327 boric acid Substances 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 235000011148 calcium chloride Nutrition 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 235000013312 flour Nutrition 0.000 description 1
- 239000006260 foam Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 125000004383 glucosinolate group Chemical group 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-M hydroxide Chemical compound [OH-] XLYOFNOQVPJJNP-UHFFFAOYSA-M 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- VTHJTEIRLNZDEV-UHFFFAOYSA-L magnesium dihydroxide Chemical compound [OH-].[OH-].[Mg+2] VTHJTEIRLNZDEV-UHFFFAOYSA-L 0.000 description 1
- 239000000347 magnesium hydroxide Substances 0.000 description 1
- 229910001862 magnesium hydroxide Inorganic materials 0.000 description 1
- 235000012254 magnesium hydroxide Nutrition 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- NBTOZLQBSIZIKS-UHFFFAOYSA-N methoxide Chemical compound [O-]C NBTOZLQBSIZIKS-UHFFFAOYSA-N 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 235000014571 nuts Nutrition 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 229940085127 phytase Drugs 0.000 description 1
- OTYBMLCTZGSZBG-UHFFFAOYSA-L potassium sulfate Chemical compound [K+].[K+].[O-]S([O-])(=O)=O OTYBMLCTZGSZBG-UHFFFAOYSA-L 0.000 description 1
- 229910052939 potassium sulfate Inorganic materials 0.000 description 1
- 239000001120 potassium sulphate Substances 0.000 description 1
- 235000011151 potassium sulphates Nutrition 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000004317 sodium nitrate Substances 0.000 description 1
- 235000010344 sodium nitrate Nutrition 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 235000011152 sodium sulphate Nutrition 0.000 description 1
- 229940001941 soy protein Drugs 0.000 description 1
- 239000004455 soybean meal Substances 0.000 description 1
- 238000001256 steam distillation Methods 0.000 description 1
- 239000004575 stone Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 235000011149 sulphuric acid Nutrition 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 108010058651 thioglucosidase Proteins 0.000 description 1
- 238000003809 water extraction Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 210000005253 yeast cell Anatomy 0.000 description 1
Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D11/00—Solvent extraction
- B01D11/02—Solvent extraction of solids
- B01D11/0288—Applications, solvents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08H—DERIVATIVES OF NATURAL MACROMOLECULAR COMPOUNDS
- C08H8/00—Macromolecular compounds derived from lignocellulosic materials
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12F—RECOVERY OF BY-PRODUCTS OF FERMENTED SOLUTIONS; DENATURED ALCOHOL; PREPARATION THEREOF
- C12F3/00—Recovery of by-products
- C12F3/10—Recovery of by-products from distillery slops
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E50/00—Technologies for the production of fuel of non-fossil origin
- Y02E50/10—Biofuels, e.g. bio-diesel
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Biochemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Botany (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- General Engineering & Computer Science (AREA)
- Gastroenterology & Hepatology (AREA)
- Biophysics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Materials Engineering (AREA)
- Polymers & Plastics (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
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 wherein the macromolecules are comprised in the dissolved solids
Description
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
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 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.
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 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.
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.
[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.
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.
[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.
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.
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.
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.
[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
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
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.
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 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.
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.
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 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 =
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.
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
(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 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.
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, wherein the macromolecules are comprised in the dissolved solids.
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.
[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.
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.
[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.
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.
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 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.
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 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
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
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.
Defatted meal was analyzed for protein and oil content.
[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 ยจ 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.
% N = (mL of 0.2N HCI sample ยจ 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.
Example 2: Extraction of mustard (Brassica juncea) protein with water (comparative example)
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
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 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.
dissolved in glycerin (a co-product of biodiesel production). After that the solution 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 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.
The mucilage was then transferred to the moisture tint and was placed in the 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
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 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 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
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 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 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.
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 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 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 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.
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-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 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 251-1499.7633 1500.8154 Cruciferin 50-61 IEVWDHHAPQLR
(SEQ ID NO: 13) 2070.0745 2071.1272 Cruciferin 420-GLPLEVISNGYQISPQEAR
(SEQ ID NO: 14) 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 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 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 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 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 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 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
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.
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-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 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 251-1499.7633 1500.8154 Cruciferin 50-61 IEVWDHHAPQLR
(SEQ ID NO: 13) 2070.0745 2071.1272 Cruciferin 420-GLPLEVISNGYQISPQEAR
(SEQ ID NO: 14) 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 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 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 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 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 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 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
Claims (31)
1. 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.
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.
2. The process of claim 1, wherein 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.
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.
3. The process of claim 2, wherein the carbohydrate rich-biomass comprises starch as a major carbohydrate.
4. The process of claim 2, wherein the carbohydrate rich-biomass comprises cellulose as a major carbohydrate.
5. The process of claim 2, wherein the carbohydrate rich-biomass comprises sucrose as a major carbohydrate.
6. The process of claim 3, wherein the carbohydrate rich-biomass is a seed.
7. The process of claim 6, wherein the carbohydrate rich-biomass is a cereal.
8. The process of claim 7, wherein the cereal is corn, wheat, rice, barley, oats or sorghum or a mixture thereof.
9. The process of claim 3, wherein the carbohydrate rich-biomass is a plant stem or tuber.
10. The process of claim 9, wherein the stem or tuber is from a potato or sweet sorghum.
11. The process of claim 3, wherein the starch is converted to glucose by one or more enzymes or catalysts to produce a fermentable sugar.
12. The process of claim 4, wherein the cellulose is depolymerized by one or more enzymes or catalysts to produce a fermentable sugar.
13. The process of any one of claims 2-12, wherein the microorganism is a yeast or a bacterium.
14. The process of any one of claims 2-13, wherein the suspended solids are removed from the thin stillage by centrifugation or sedimentation.
15. The process of any one of claim 2-14, wherein the macromolecular solutes are removed from the thin stillage by ultrafiltration or nanofiltration.
16. The process of any one of claims 1-15, wherein the pH of the thin stillage is increased above pH 8.
17. The process according of claim 16, wherein the pH of the thin stillage is adjusted above pH8 using waste alkaline solution from biodiesel production.
18. The process of claim 17, wherein the waste alkaline solution from biodiesel production is a glycerol/potassium hydroxide solution.
19. The process of any one of claims 1-16, wherein the pH of the thin stillage is decreased below pH 5.
20. The process of any one of claims 1-19, wherein the thin stillage further comprises an additive.
21. The process of claim 20, wherein the additive is a salt, detergent or zwitterions or a mixture thereof.
22. The process of any one of claims 1-21, wherein the slurry is heated.
23. The process of any one of claims 1-22, wherein the undissolved solids are separated by filtration or centrifugation.
24. The process of any one of claims 1-23, wherein the dissolved solids are isolated by ultrafiltration, diafiltration, reverse osmosis or nanofiltration.
25. The process of any one of claims 1-23, wherein the dissolved solids are isolated by evaporation.
26. The process of any one of claims 1-23, wherein the dissolved solids are isolated by precipitation.
27. The process of claim 26, wherein the dissolved solids are precipitated by a change in pH or ionic strength.
28. The process of any one of claims 1-27, wherein the contacting of the biomass material with the thin stillage is conducted in a continuous process.
29. The process of any one of claims 1-27, wherein the contacting of the biomass material with the thin stillage is conducted in a batch process.
30. The process of any one of claims 1-27, wherein the contacting of the biomass material with the thin stillage is conducted in a countercurrent process.
31. The process of any one of claims 2-15, wherein 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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