CN113087760A - Method for extracting protein from vinasse - Google Patents
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- CN113087760A CN113087760A CN202110432149.7A CN202110432149A CN113087760A CN 113087760 A CN113087760 A CN 113087760A CN 202110432149 A CN202110432149 A CN 202110432149A CN 113087760 A CN113087760 A CN 113087760A
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- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 69
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 69
- 238000000034 method Methods 0.000 title claims abstract description 59
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 183
- 239000007788 liquid Substances 0.000 claims abstract description 86
- 238000000855 fermentation Methods 0.000 claims abstract description 68
- 230000004151 fermentation Effects 0.000 claims abstract description 68
- 238000002156 mixing Methods 0.000 claims abstract description 65
- 239000007787 solid Substances 0.000 claims abstract description 56
- 150000002772 monosaccharides Chemical class 0.000 claims abstract description 24
- 238000000926 separation method Methods 0.000 claims abstract description 24
- 239000002253 acid Substances 0.000 claims abstract description 23
- 235000019750 Crude protein Nutrition 0.000 claims abstract description 20
- 230000008569 process Effects 0.000 claims abstract description 20
- 238000001035 drying Methods 0.000 claims abstract description 19
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 33
- 102000004190 Enzymes Human genes 0.000 claims description 32
- 108090000790 Enzymes Proteins 0.000 claims description 32
- 229940088598 enzyme Drugs 0.000 claims description 32
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 26
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 22
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 18
- 239000002994 raw material Substances 0.000 claims description 17
- 238000010306 acid treatment Methods 0.000 claims description 16
- 239000002002 slurry Substances 0.000 claims description 15
- 241000894006 Bacteria Species 0.000 claims description 13
- 108010059892 Cellulase Proteins 0.000 claims description 13
- 229940106157 cellulase Drugs 0.000 claims description 13
- 238000009210 therapy by ultrasound Methods 0.000 claims description 13
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 12
- 102000004139 alpha-Amylases Human genes 0.000 claims description 12
- 108090000637 alpha-Amylases Proteins 0.000 claims description 12
- 229940024171 alpha-amylase Drugs 0.000 claims description 12
- 229910052757 nitrogen Inorganic materials 0.000 claims description 11
- 230000002255 enzymatic effect Effects 0.000 claims description 9
- 239000000413 hydrolysate Substances 0.000 claims description 9
- 239000002904 solvent Substances 0.000 claims description 8
- 241000228212 Aspergillus Species 0.000 claims description 7
- PAWQVTBBRAZDMG-UHFFFAOYSA-N 2-(3-bromo-2-fluorophenyl)acetic acid Chemical compound OC(=O)CC1=CC=CC(Br)=C1F PAWQVTBBRAZDMG-UHFFFAOYSA-N 0.000 claims description 6
- 101710121765 Endo-1,4-beta-xylanase Proteins 0.000 claims description 6
- 241000235527 Rhizopus Species 0.000 claims description 6
- 241000588902 Zymomonas mobilis Species 0.000 claims description 6
- 238000000605 extraction Methods 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 6
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 claims description 4
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 4
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 4
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 4
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 4
- 239000004202 carbamide Substances 0.000 claims description 4
- 238000006243 chemical reaction Methods 0.000 claims description 4
- 229910017604 nitric acid Inorganic materials 0.000 claims description 4
- 230000007071 enzymatic hydrolysis Effects 0.000 claims description 3
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 claims description 3
- 238000011081 inoculation Methods 0.000 claims description 3
- 238000002360 preparation method Methods 0.000 claims description 3
- 230000009286 beneficial effect Effects 0.000 abstract description 5
- 239000006227 byproduct Substances 0.000 abstract description 3
- 238000011161 development Methods 0.000 abstract description 2
- 102100034613 Annexin A2 Human genes 0.000 abstract 1
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- 239000000243 solution Substances 0.000 description 69
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 18
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 16
- 240000008042 Zea mays Species 0.000 description 15
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 description 15
- 235000002017 Zea mays subsp mays Nutrition 0.000 description 15
- 235000005822 corn Nutrition 0.000 description 15
- 238000001914 filtration Methods 0.000 description 13
- 238000004108 freeze drying Methods 0.000 description 11
- 238000000751 protein extraction Methods 0.000 description 11
- 235000013339 cereals Nutrition 0.000 description 10
- 238000001816 cooling Methods 0.000 description 10
- 239000000843 powder Substances 0.000 description 10
- 230000001954 sterilising effect Effects 0.000 description 10
- 239000002054 inoculum Substances 0.000 description 9
- 229920002261 Corn starch Polymers 0.000 description 7
- 230000000052 comparative effect Effects 0.000 description 7
- 239000008120 corn starch Substances 0.000 description 7
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 6
- 229920002472 Starch Polymers 0.000 description 6
- 235000019698 starch Nutrition 0.000 description 5
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- 238000004659 sterilization and disinfection Methods 0.000 description 5
- 239000000835 fiber Substances 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 238000007710 freezing Methods 0.000 description 3
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- 239000000446 fuel Substances 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- BDERNNFJNOPAEC-UHFFFAOYSA-N propan-1-ol Chemical compound CCCO BDERNNFJNOPAEC-UHFFFAOYSA-N 0.000 description 3
- 102000005741 Metalloproteases Human genes 0.000 description 2
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- 230000008901 benefit Effects 0.000 description 2
- 229920002678 cellulose Polymers 0.000 description 2
- 239000001913 cellulose Substances 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
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- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 235000000346 sugar Nutrition 0.000 description 2
- 150000008163 sugars Chemical class 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 241000228245 Aspergillus niger Species 0.000 description 1
- 240000006439 Aspergillus oryzae Species 0.000 description 1
- 235000002247 Aspergillus oryzae Nutrition 0.000 description 1
- 101710089042 Demethyl-4-deoxygadusol synthase Proteins 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 229920002488 Hemicellulose Polymers 0.000 description 1
- 238000007696 Kjeldahl method Methods 0.000 description 1
- 240000007594 Oryza sativa Species 0.000 description 1
- 235000007164 Oryza sativa Nutrition 0.000 description 1
- 241000235342 Saccharomycetes Species 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 240000006394 Sorghum bicolor Species 0.000 description 1
- 235000011684 Sorghum saccharatum Nutrition 0.000 description 1
- 241000209140 Triticum Species 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 239000003929 acidic solution Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 description 1
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000002485 combustion reaction Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000002803 fossil fuel Substances 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 229920005610 lignin Polymers 0.000 description 1
- 239000012978 lignocellulosic material Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 238000000053 physical method Methods 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 239000011241 protective layer Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 239000008213 purified water Substances 0.000 description 1
- 235000009566 rice Nutrition 0.000 description 1
- 229940100486 rice starch Drugs 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- JBJWASZNUJCEKT-UHFFFAOYSA-M sodium;hydroxide;hydrate Chemical compound O.[OH-].[Na+] JBJWASZNUJCEKT-UHFFFAOYSA-M 0.000 description 1
- 230000001360 synchronised effect Effects 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 238000010792 warming Methods 0.000 description 1
- 229940100445 wheat starch Drugs 0.000 description 1
- 229920001221 xylan Polymers 0.000 description 1
- 150000004823 xylans Chemical class 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/145—Extraction; Separation; Purification by extraction or solubilisation
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/02—Monosaccharides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/20—Preparation of compounds containing saccharide radicals produced by the action of an exo-1,4 alpha-glucosidase, e.g. dextrose
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
- C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
- C12P7/06—Ethanol, i.e. non-beverage
- C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
-
- 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
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/59—Biological synthesis; Biological purification
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- Chemical & Material Sciences (AREA)
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Biotechnology (AREA)
- Analytical Chemistry (AREA)
- Biophysics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to the technical field of renewable energy sources and bioengineering, and discloses a method for extracting protein from vinasse. The invention provides a method for extracting protein from vinasse, which comprises the following steps: (1) carrying out acid pretreatment, enzymolysis and solid-liquid separation I on the vinasse to obtain solid containing protein and enzymolysis liquid containing monosaccharide; (2) mixing the solid containing the protein with organic alcohol and strong base, performing solid-liquid separation II to obtain mixed clear liquid, adjusting the pH of the mixed clear liquid to acidity, performing solid-liquid separation III to obtain crude protein solid, and drying the crude protein solid to obtain the protein I. The method provided by the invention can effectively promote the release of protein and monosaccharide, can improve the resource utilization rate of fermentation byproducts, and is beneficial to the development of a high-valued utilization process of vinasse.
Description
Technical Field
The invention relates to the technical field of renewable energy sources and bioengineering, in particular to a method for extracting protein from vinasse.
Background
The demand for fuels is rapidly increased in the current times, and the exploitation and combustion of fossil fuels can cause serious pollution to the environment, which causes the problems of global warming, sea level rising, biodiversity threatened and the like. The fuel ethanol is a renewable resource with great prospect, can replace part of gasoline, and the production of the fuel ethanol in China mainly takes starchy grains as raw materials, ethanol is extracted by starch liquefaction, synchronous saccharification and distillation, and a large amount of by-products, namely vinasse, can be obtained at the same time.
The vinasse is rich in components, comprises crude fiber with high content, crude ash, crude protein, cellulose, xylan, arabinose, residual starch and the like, and has great research significance and wide application prospect in the field of corn ethanol. In recent years, lees are mainly used in the field of feed, and feed is obtained by mixing concentrated liquid and partially dried solid after solid-liquid separation of lees and then continuously drying, but the economic improvement of the ethanol process is not large.
The lignocellulose in the vinasse is reasonably developed and utilized, so that the utilization value of the lignocellulose can be remarkably improved. The complex structure of lignocellulosic material is a degradation-resistant barrier that plants have evolved over the billions to protect themselves from attack by microorganisms and their enzyme systems, with cellulose tightly enclosed in a protective layer of lignin and hemicellulose, among other things. The degradability of the corn ethanol is obviously improved after proper pretreatment, the corn ethanol is converted into fermentable sugars by cellulase, and the sugars are converted into ethanol by ethanol zymophyte, so that the economic benefit of the corn ethanol process is improved. At present, there are many protein extraction methods, such as mechanical grinding, ultrasound, microwave and other physical methods, and alcohol, alkaline, alcohol-alkaline and other chemical methods, but most of the methods are single, strict in operation procedure, complex in extraction conditions and the like, and are not favorable for improving ethanol production efficiency and economy.
Disclosure of Invention
The invention aims to overcome the problems in the prior art and provide a method for extracting protein from vinasse, which can promote the release of protein and monosaccharide and is beneficial to improving the economic benefit of the ethanol industry.
In order to achieve the above object, the present invention provides a method for extracting protein from distiller's grains, comprising the steps of:
(1) carrying out acid pretreatment, enzymolysis and solid-liquid separation I on the vinasse to obtain solid containing protein and enzymolysis liquid containing monosaccharide;
(2) mixing and extracting the solid containing the protein with organic alcohol and strong base, performing solid-liquid separation II to obtain mixed clear liquid, adjusting the pH of the mixed clear liquid to acidity, performing solid-liquid separation III to obtain crude protein solid, and drying the crude protein solid I to obtain the protein.
Preferably, in the step (1), the preparation method of the vinasse comprises the following steps: separating ethanol from ethanol fermentation liquor to obtain fermented mash, performing solid-liquid separation IV on the fermented mash to obtain wet cake and fermented clear liquid, concentrating the fermented clear liquid to obtain fermented thick slurry, mixing the fermented thick slurry and the wet cake, and drying II.
Preferably, the method for preparing the ethanol fermentation liquor comprises the following steps: mixing a starchy raw material, alpha-amylase and a liquefying solvent for liquefaction, and then mixing the mixture with saccharifying enzyme, a nitrogen source and ethanol zymocyte I for fermentation I; wherein the nitrogen source is selected from one or more of urea, ammonium sulfate and ammonium nitrate, and the ethanol fermentation bacteria I is selected from one or more of yeast, zymomonas mobilis, aspergillus and rhizopus.
Preferably, the amount of the alpha-amylase is 0.01-0.05g, the amount of the saccharifying enzyme is 0.01-0.05g, the amount of the nitrogen source is 0.05-0.3g, and the amount of the liquefying solvent is 150-300g, relative to 100g of the dry weight of the starchy raw material; the ethanol isThe inoculation amount of zymocyte I is 1 multiplied by 108-5×108cfu/mL；
Preferably, the condition for liquefaction is at least satisfied: the pH is 3.5-4.5, the temperature is 75-95 ℃, and the time is 2-5 h;
the condition of fermentation I at least satisfies: the temperature is 25-35 ℃, the rotation speed is 150-.
Preferably, in step (1), the acid pretreatment comprises: mixing the vinasse with water, mixing the vinasse with an acid solution to obtain a pretreatment solution, and performing acid treatment on the pretreatment solution;
preferably, the mass ratio of the vinasse to the water is 1: 1-19;
the acid solution is selected from one or more of sulfuric acid solution, hydrochloric acid solution and nitric acid solution, and the concentration of the acid in the pretreatment solution is 0.5-3 wt%;
the acid treatment conditions at least satisfy: the temperature is 100 ℃ and 130 ℃, and the time is 30-90 min.
Preferably, in step (1), the enzymatic hydrolysis process includes: mixing the pretreatment solution after acid treatment with enzyme and then reacting;
preferably, the enzyme is selected from one or more of saccharifying enzyme, cellulase and xylanase, and the mass ratio of the enzyme I to the pretreatment solution after acid treatment is 0.05-1.4: 100, respectively;
the reaction conditions at least satisfy: the pH value is 4-5.5, the temperature is 45-55 ℃, the rotation speed is 200-300rpm, and the time is 30-64 h.
Preferably, in the step (2), the amount of the organic alcohol is less than or equal to 90g, and the amount of the strong base is less than or equal to 5g, relative to 10g of the protein-containing solid by dry weight;
the process of mixed extraction comprises the following steps: mixing the solid containing the protein with organic alcohol and strong base, and then carrying out ultrasonic treatment, wherein the ultrasonic treatment at least meets the following conditions: the temperature is 20-70 deg.C, and the time is 20-120 min.
Preferably, in the step (2), the pH of the mixed clear solution is adjusted to 4 to 5.
Preferably, the method further comprises: and (2) mixing the monosaccharide-containing enzymatic hydrolysate obtained in the step (1) with ethanol fermentation bacteria II to carry out fermentation II.
Preferably, the ethanol fermentation bacteria II is selected from one or more of yeast, zymomonas mobilis, aspergillus and rhizopus;
the conditions of fermentation II at least satisfy: the temperature is 25-35 ℃, the rotating speed is 150-8-5×108cfu/mL。
Through the technical scheme, the invention has the beneficial effects that:
(1) when the method utilizes the vinasse to extract the protein, the vinasse is subjected to acid method pretreatment and enzyme hydrolysis treatment, so that the complex structure of mutual combination among starch, fiber and protein in the vinasse is effectively broken, the release of the protein is promoted, the subsequent protein extraction is facilitated, and the vinasse subjected to acid method pretreatment is subjected to enzyme hydrolysis treatment to obtain monosaccharide-rich enzymatic hydrolysate;
(2) the invention uses the vinasse as the raw material to extract the protein, can further improve the added value and the resource utilization rate of the fermentation by-products, and is beneficial to the development of a high-valued utilization process of the vinasse;
(3) in the invention, the enzymolysis liquid containing monosaccharide after protein extraction is directly used as the culture medium for ethanol fermentation, so that the cost of the ethanol process can be effectively saved, and the economy of the whole ethanol process is improved.
Detailed Description
The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.
The invention provides a method for extracting protein from vinasse, which comprises the following steps:
(1) carrying out acid pretreatment, enzymolysis and solid-liquid separation I on the vinasse to obtain solid containing protein and enzymolysis liquid containing monosaccharide;
(2) mixing and extracting the solid containing the protein with organic alcohol and strong base, performing solid-liquid separation II to obtain mixed clear liquid, adjusting the pH of the mixed clear liquid to acidity, performing solid-liquid separation III to obtain crude protein solid, and drying the crude protein solid I to obtain the protein.
According to the invention, the solid-liquid separation I, the solid-liquid separation II and the solid-liquid separation III can respectively adopt conventional separation modes such as filtration, centrifugation and the like, and the solid-liquid separation I and the solid-liquid separation II can adopt the same mode or different modes; the drying I can adopt any concentration drying mode such as freeze drying, microwave drying or vacuum heating drying.
According to the invention, the Distillers Grains refer to Dried Distillers Grains with soluble solids, preferably the Distillers Dried Grains with Solubles. Specifically, in the step (1), the preparation method of the vinasse comprises the following steps: separating ethanol from ethanol fermentation liquor to obtain fermented mash, performing solid-liquid separation IV on the fermented mash to obtain wet cake and fermented clear liquid, concentrating the fermented clear liquid to obtain fermented thick slurry, mixing the fermented thick slurry with the wet cake, and drying II to obtain the DDGS feed.
According to the invention, the solid-liquid separation IV can adopt conventional separation modes such as filtration and centrifugation, and the drying II can adopt any one of concentration drying modes such as freeze drying, microwave drying or vacuum heating drying; the concentrated fermentation slurry obtained by concentrating the clear fermentation liquid can be obtained by concentrating a part of clear fermentation liquid and mixing the concentrated fermentation liquid with the clear fermentation liquid which is not concentrated, or can be obtained by concentrating the whole clear fermentation liquid.
According to the present invention, the method for obtaining the ethanol fermentation liquid is not particularly limited, and for example, the ethanol fermentation liquid may be obtained by culturing ethanol fermentation bacteria using corn as a raw material, or may be obtained by culturing ethanol fermentation bacteria using rice, wheat, sorghum, or other crops as a raw material. Preferably, the method for preparing the ethanol fermentation liquor comprises the following steps: mixing a starchy raw material, alpha-amylase and a liquefying solvent for liquefying, and then mixing the mixture with saccharifying enzyme, a nitrogen source and ethanol fermentation bacteria I for fermentation I.
According to the invention, the starchy raw material can be a grain raw material or a potato raw material, and specifically can be a corn starch raw material, a wheat starch raw material, a rice starch raw material and the like; the liquefaction solvent and the enzymolysis solvent can be water respectively, and specifically can be purified water, distilled water or tap water.
According to the invention, the nitrogen source can adopt conventional nitrogen source substances, and preferably, the nitrogen source is selected from one or more of urea, ammonium sulfate and ammonium nitrate so as to be matched with saccharifying enzyme, promote the fermentation rate of ethanol zymophyte I and improve the yield of ethanol.
According to the invention, the ethanol fermentation bacteria I can adopt any strain capable of producing ethanol through fermentation, and preferably, the ethanol fermentation bacteria I is selected from one or more of saccharomycetes, zymomonas mobilis, aspergillus and rhizopus. Illustratively, the yeast may be Saccharomyces cerevisiae, and the Aspergillus may be Aspergillus niger or Aspergillus oryzae.
According to the invention, relative to 100g of dry weight of the starchy raw material, the dosage of the alpha-amylase is 0.01-0.05g, the dosage of the saccharifying enzyme is 0.01-0.05g, the dosage of the nitrogen source is 0.05-0.3g, and the dosage of the liquefying solvent is 150-300 g; the inoculation amount of the ethanol zymocyte I is 1 multiplied by 108-5×108cfu/mL. The inventors have found that in this preferred embodiment, ethanol production can be effectively increased and an ethanol fermentation broth rich in crude fiber, crude ash, crude protein and a small amount of starch can be obtained.
According to the invention, the conditions of liquefaction are at least satisfied: a pH of 3.5 to 4.5, specifically, 3.5, 3.7, 3.9, 4.1, 4.3, 4.5, or any value in the range of any two of these values; the temperature is 75-95 ℃, and specifically can be 75 ℃, 80 ℃, 85 ℃, 90 ℃, 95 ℃ or any value in the range formed by any two of the values; the time is 2 to 5 hours, and specifically, may be 2 hours, 3 hours, 4 hours, 5 hours, or any value in a range formed by any two of these values.
According to the invention, the conditions of the fermentation I can be selected conventionally in the field, and the conditions such as temperature, time and the like required by the growth of the ethanol zymocyte I can be provided. Preferably, the conditions of fermentation I at least satisfy: the temperature is 25-35 deg.C, specifically 25 deg.C, 27 deg.C, 29 deg.C, 31 deg.C, 33 deg.C, 35 deg.C, or any value in the range of any two of these values; the rotation speed is 150-300rpm, specifically any value in the range of 150rpm, 180rpm, 210rpm, 240rpm, 270rpm, 300rpm and any two of these values; the time is 60 to 85h, and specifically, may be any value in a range of 60h, 65h, 70h, 75h, 80h, 85h, or any two of these values.
According to the present invention, in the step (1), the acid pretreatment process comprises: and mixing the vinasse with water, mixing the vinasse with an acid solution to obtain a pretreatment solution, and performing acid treatment on the pretreatment solution. The acid solution may be a conventional acidic solution, and specifically may be a solution containing a strong acid such as hydrochloric acid, sulfuric acid, and nitric acid, or may be a solution containing a weak acid such as acetic acid and citric acid. Preferably, the vinasse-water solution is subjected to acid treatment by adopting a solution containing strong acid, so that the complex structure of mutual combination among starch, fiber and protein in the vinasse is broken through enzymolysis, and the release of the protein is promoted.
According to the invention, the mass ratio of the vinasse to the water is 1: 1-19; the acid solution is selected from one or more of sulfuric acid solution, hydrochloric acid solution and nitric acid solution, and the concentration of the acid in the pretreatment solution is 0.5-3 wt%; specifically, it may be any value within a range of 0.5 wt%, 1 wt%, 1.5 wt%, 2 wt%, 2.5 wt%, 3 wt%, or any two of these values. The inventors have found that in this preferred embodiment, the acid treatment of the aqueous distillers grain solution is more effective, which is beneficial to promote the efficiency of the enzymatic hydrolysis.
According to the present invention, the conditions of the acid treatment may be chosen conventionally in the art, and preferably, the conditions of the acid treatment at least satisfy: the temperature is 100 ℃ or 130 ℃, and specifically, it may be 100 ℃, 105 ℃, 110 ℃, 115 ℃, 120 ℃, 125 ℃, 130 ℃, or any value in the range formed by any two of these values; the time is 30-90min, specifically 30min, 40min, 50min, 60min, 70min, 80min, 90min, and any value in the range formed by any two of these values.
According to the invention, in the step (1), the enzymolysis process comprises the following steps: mixing the pretreatment solution after acid treatment with enzyme and then reacting; preferably, the enzyme is selected from one or more of saccharifying enzyme, cellulase and xylanase, and the mass ratio of the enzyme I to the pretreatment solution after acid treatment is 0.05-1.4: 100. the inventors have found that in this preferred embodiment, it is advantageous to increase the yield and efficiency of protein extraction from acid pretreated whole stillage.
According to the present invention, the conditions of the reaction may be chosen as is conventional in the art, preferably at least the conditions of the reaction are such that: a pH of 4 to 5.5, specifically 4, 4.3, 4.6, 4.9, 5.2, 5.5, and any value in the range of any two of these values; the temperature is 45-55 deg.C, specifically 45 deg.C, 47 deg.C, 49 deg.C, 51 deg.C, 53 deg.C, 55 deg.C, or any two of these values; the rotation speed is 200-300rpm, and specifically may be any value in the range of 200rpm, 220rpm, 240rpm, 260rpm, 280rpm, 300rpm and any two of these values; the time is 30 to 64h, and specifically, may be any value in a range of 30h, 35h, 40h, 45h, 50h, 55h, 60h, 64h, or any two of these values.
According to the invention, in the step (2), the solid containing the protein can be directly mixed with the organic alcohol and the strong base, or the solid containing the protein can be firstly mixed with water to form a protein solid-water solution, and then the protein solid-water solution is mixed with the organic alcohol and the strong base; the organic alcohol is preferably C1-C4 organic alcohol, and specifically can be one or any combination of methanol, ethanol and n-propanol, wherein the organic alcohol can be added in the form of pure organic alcohol or organic alcohol-water solution; the strong base can be sodium hydroxide and/or potassium hydroxide, and can be added in the form of solid particles or strong base-water solution.
According to the invention, the organic alcohol is used in an amount less than or equal to 90g and the strong base is used in an amount less than or equal to 5g, relative to 10g of the protein-containing solid on a dry weight basis. Preferably, at least one of the solid containing protein, the organic alcohol and the strong base is added in the form of an aqueous solution, so that after the solid containing protein, the organic alcohol, the strong base and water are mixed, the mass fraction of the solid containing protein is 10-30 wt%, the mass fraction of the organic alcohol is less than 90 wt%, and the mass fraction of the strong base is less than 5 wt%. The inventors have found that in this preferred embodiment it is advantageous to increase the efficiency and yield of purification of the protein in the protein-containing solid.
According to the invention, the process of hybrid extraction comprises: mixing the solid containing the protein with organic alcohol and strong base, and then carrying out ultrasonic treatment, wherein the ultrasonic treatment at least meets the following conditions: the temperature is 20-70 deg.C, specifically 20 deg.C, 30 deg.C, 40 deg.C, 50 deg.C, 60 deg.C, 70 deg.C, or any value in the range of any two of these values; the time is 20-120min, and specifically may be 20min, 40min, 60min, 80min, 100min, 120min, or any value in the range of any two of these values.
According to the invention, in step (2), the pH of the mixed clear solution is adjusted to 4 to 5, and specifically may be any value in the range of 4, 4.2, 4.4, 4.6, 4.8, 5 and any two of these values.
The inventor finds in the research process that the monosaccharide-containing enzymatic hydrolysate obtained in step (1) of the method for extracting protein from distiller's grains can provide nutrient elements required in the growth process of the strain for ethanol fermentation, and can be further used as a culture medium for ethanol fermentation. Specifically, the method further comprises: mixing the monosaccharide-containing enzymatic hydrolysate obtained in the step (1) with ethanol fermentation bacteria II to carry out fermentation II; wherein, the monosaccharide-containing enzymolysis liquid needs to be sterilized for fermentation II, and ethanol can be collected after the fermentation II is finished. Under the preferred embodiment, the production cost of the ethanol process can be effectively saved, and the economy of the whole ethanol process is improved.
According to the invention, the ethanol fermentation bacteria II is selected from one or more of yeast, zymomonas mobilis, aspergillus and rhizopus; the conditions of fermentation II at least satisfy: the temperature is 25-35 ℃, the rotating speed is 150-8-5 ×108cfu/mL。
The present invention will be described in detail below by way of examples.
In the following examples, the protein content was measured by Kjeldahl method (GB/T5009.5-2010) and the ethanol concentration was measured by high performance liquid chromatography (analysis conditions: HPX-87H column, differential detector, mobile phase 0.005M H2SO4The flow rate is 0.6mL/min, the column temperature is 65 ℃, and the time is 25 min); the saccharomyces cerevisiae is from Angel Yeast, corn starch from China oil and fat food group, alpha-amylase from Jenenergy Ke (China) bioengineering, saccharifying enzyme from Shandong Longmao Bio, cellulase and xylanase from Qingdao blue Bio, and other raw materials and reagents are commercially available.
Example 1
(1) Mixing 1000g of corn starch in dry weight with a certain amount of water to reach a dry matter concentration of 30%, adding 0.12g of alpha-amylase and adjusting pH to 4.3-4.4, liquefying at 89 deg.C for 3h to obtain corn liquefied liquid, adjusting pH of the corn liquefied liquid to 3.8-4.0, adding 0.325g of saccharifying enzyme and 1.2g of urea, and performing liquefaction at 2.5 × 108Inoculating the cfu/mL inoculum size into saccharomyces cerevisiae, and fermenting for 72h under the conditions that the temperature is 30 ℃ and the rotating speed is 250rpm to obtain ethanol fermentation liquor;
(2) distilling the ethanol fermentation liquor obtained in the step (1) to remove ethanol to obtain fermented mash, filtering the fermented mash to obtain wet cake and fermented clear liquor, concentrating a part of the fermented clear liquor to obtain fermented thick slurry, mixing the fermented thick slurry and the wet cake, and then freezing and drying to obtain vinasse;
(3) mixing 40g of the vinasse obtained in the step (2) with 160g of water, mixing with a sulfuric acid solution to obtain a pretreatment solution, enabling the mass concentration of sulfuric acid in the pretreatment solution to be 2.0 wt%, reacting for 60min at 120 ℃ in a sterilization pot, cooling, adjusting the pH to be 5.0, adding saccharifying enzyme and cellulase (the mass ratio of the saccharifying enzyme to the cellulase to the pretreatment solution is 0.1: 0.6: 100), reacting for 48h at 50 ℃ and 250rpm, and centrifuging to obtain a solid containing protein and an enzymolysis solution containing monosaccharide;
(4) mixing 10g of the solid containing the protein obtained in the step (3) with 90g of water, uniformly mixing with 50g of ethanol and 5g of sodium hydroxide, carrying out ultrasonic treatment at 45 ℃ for 60min, filtering to obtain a mixed clear liquid, cooling the mixed clear liquid, adjusting the pH value to 4.8, centrifuging to collect a crude protein solid, and carrying out reduced pressure concentration and freeze drying on the crude protein solid to obtain protein powder;
(5) sterilizing the enzymolysis solution containing monosaccharide obtained in step (3), and adding 2.5 × 108Inoculating the cfu/mL inoculum size into saccharomyces cerevisiae, and fermenting for 72h under the conditions that the temperature is 30 ℃ and the rotating speed is 250rpm to obtain the enzymolysis liquid fermentation liquid.
Example 2
(1) Mixing 1000g of corn starch with a certain amount of water to dry matter concentration of 25%, adding 0.1g of alpha-amylase and adjusting pH to 3.8-4.0, liquefying at 75 deg.C for 5 hr to obtain corn liquefied liquid, adjusting pH of the corn liquefied liquid to 3.8-4.0, adding 0.5g of saccharifying enzyme and 0.5g of ammonium sulfate, and performing liquefaction at 1 × 108Inoculating the cfu/mL inoculum size into saccharomyces cerevisiae, and fermenting for 60h under the conditions that the temperature is 35 ℃ and the rotating speed is 300rpm to obtain ethanol fermentation liquor;
(2) distilling the ethanol fermentation liquor obtained in the step (1) to remove ethanol to obtain fermented mash, filtering the fermented mash to obtain wet cake and fermented clear liquor, concentrating all the fermented clear liquor to obtain fermented thick slurry, mixing the fermented thick slurry with the wet cake, and freeze-drying to obtain vinasse;
(3) mixing 30g of the vinasse obtained in the step (2) with 170g of water, mixing with a concentrated hydrochloric acid solution to obtain a pretreatment solution, enabling the mass concentration of hydrochloric acid in the pretreatment solution to be 0.5 wt%, reacting for 90min at 100 ℃ in a sterilization pot, cooling, adjusting the pH to be 5.5, adding saccharifying enzyme and xylanase (the mass ratio of the saccharifying enzyme to the xylanase to the pretreatment solution is 0.7: 0.7: 100), reacting for 64h at 45 ℃ and 200rpm, and centrifuging to obtain a solid containing protein and an enzymolysis solution containing monosaccharide;
(4) mixing 10g of the solid containing the protein obtained in the step (3) with 20g of water, uniformly mixing the solid with 90g of ethanol and 5g of potassium hydroxide, carrying out ultrasonic treatment at the temperature of 20 ℃ for 120min, filtering to obtain a mixed clear liquid, cooling the mixed clear liquid, adjusting the pH value to 4.7, centrifuging to collect a crude protein solid, and carrying out reduced pressure concentration and freeze drying on the crude protein solid to obtain protein powder;
(5) sterilizing the monosaccharide-containing enzymatic hydrolysate obtained in the step (3), and then performing enzymolysis at a ratio of 1 × 108Inoculating the cfu/mL inoculum size into saccharomyces cerevisiae, and fermenting for 60h under the conditions that the temperature is 35 ℃ and the rotating speed is 300rpm to obtain the enzymolysis liquid fermentation liquid.
Example 3
(1) Mixing 1000g of corn starch in dry weight with a certain amount of water until the dry matter concentration is 40%, adding 0.5g of alpha-amylase and adjusting the pH value to 4.3-4.5, liquefying at 95 ℃ for 2h to obtain a corn liquefied solution, adjusting the pH value of the corn liquefied solution to 4.3-4.5, adding 0.1g of saccharifying enzyme and 3g of ammonium nitrate, and inoculating with an amount of 5 × 108Inoculating the cfu/mL inoculum size into saccharomyces cerevisiae, and fermenting for 85h under the conditions that the temperature is 25 ℃ and the rotating speed is 150rpm to obtain ethanol fermentation liquor;
(2) distilling the ethanol fermentation liquor obtained in the step (1) to remove ethanol to obtain fermented mash, filtering the fermented mash to obtain wet cake and fermented clear liquor, concentrating a part of the fermented clear liquor to obtain fermented thick slurry, mixing the fermented thick slurry and the wet cake, and then freezing and drying to obtain vinasse;
(3) mixing 30g of the vinasse obtained in the step (2) with 300g of water, mixing with a sulfuric acid solution to obtain a pretreatment solution, enabling the mass concentration of sulfuric acid in the pretreatment solution to be 3 wt%, reacting for 30min at 130 ℃ in a sterilization pot, cooling, adjusting the pH to be 4, adding cellulase (the mass ratio of the cellulase to the pretreatment solution is 0.05: 100), reacting for 30h at 55 ℃ and 300rpm, and centrifuging to obtain a solid containing protein and an enzymolysis solution containing monosaccharide;
(4) mixing 10g of the solid containing the protein obtained in the step (3) with 40g of water, uniformly mixing with 50g of ethanol and 3g of sodium hydroxide, carrying out ultrasonic treatment at 70 ℃ for 20min, filtering to obtain a mixed clear liquid, cooling the mixed clear liquid, adjusting the pH to 4.9, centrifuging to collect a crude protein solid, and carrying out reduced pressure concentration and freeze drying on the crude protein solid to obtain protein powder;
(5) sterilizing the monosaccharide-containing enzymatic hydrolysate obtained in the step (3), and then performing enzymolysis at a ratio of 5 × 108Inoculating the cfu/mL inoculum size into saccharomyces cerevisiae, and fermenting for 85h under the conditions that the temperature is 25 ℃ and the rotating speed is 150rpm to obtain the enzymolysis liquid fermentation liquid.
Example 4
Protein extraction was carried out in the same manner as in example 3 except that the sulfuric acid concentration in the pretreatment liquid in the step (3) was 5% by weight.
Example 5
Protein extraction was carried out in the same manner as in example 3, except that the pretreatment solution in the step (3) was subjected to the acid treatment in the following manner: reacting at 85 deg.C for 25min in a sterilizing pot.
Example 6
(1) Mixing 1000g of corn starch in dry weight with a certain amount of water until the dry matter concentration is 40%, adding 0.5g of alpha-amylase and adjusting the pH value to 4.3-4.5, liquefying at 95 ℃ for 2h to obtain a corn liquefied solution, adjusting the pH value of the corn liquefied solution to 4.3-4.5, adding 0.1g of saccharifying enzyme and 3g of ammonium nitrate, and inoculating with an amount of 5 × 108Inoculating the cfu/mL inoculum size into saccharomyces cerevisiae, and fermenting for 85h under the conditions that the temperature is 25 ℃ and the rotating speed is 150rpm to obtain ethanol fermentation liquor;
(2) distilling the ethanol fermentation liquor obtained in the step (1) to remove ethanol to obtain fermented mash, filtering the fermented mash to obtain wet cake and fermented clear liquor, concentrating a part of the fermented clear liquor to obtain fermented thick slurry, mixing the fermented thick slurry and the wet cake, and then freezing and drying to obtain vinasse;
(3) mixing 20g of the vinasse obtained in the step (2) with 380g of water, mixing with a sulfuric acid solution to obtain a pretreatment solution, enabling the mass concentration of sulfuric acid in the pretreatment solution to be 0.6 wt%, reacting for 30min at 130 ℃ in a sterilization pot, cooling, adjusting the pH to be 4, adding metalloproteinases (the mass ratio of the metalloproteinases to the pretreatment solution is 0.2: 100), reacting for 40h at the temperature of 55 ℃ and the rotating speed of 300rpm, and centrifuging to obtain a solid containing proteins and an enzymolysis solution containing monosaccharides;
(4) mixing 10g of the solid containing the protein obtained in the step (3) with 40g of water, uniformly mixing with 50g of propanol and 5g of sodium hydroxide, carrying out ultrasonic treatment at 70 ℃ for 20min, filtering to obtain a mixed clear liquid, cooling the mixed clear liquid, adjusting the pH to 5.5, centrifuging to collect a crude protein solid, and carrying out reduced pressure concentration and freeze drying on the crude protein solid to obtain protein powder;
(5) sterilizing the monosaccharide-containing enzymatic hydrolysate obtained in the step (3), and then performing enzymolysis at a ratio of 5 × 108Inoculating the cfu/mL inoculum size into saccharomyces cerevisiae, and fermenting for 85h under the conditions that the temperature is 25 ℃ and the rotating speed is 120rpm to obtain the enzymolysis liquid fermentation liquid.
Example 7
Protein extraction was performed according to the method of example 3, except that the step (3) was: and (3) mixing 30g of the vinasse obtained in the step (2) with 300g of water, mixing with a sulfuric acid solution to obtain a pretreatment solution, enabling the mass concentration of sulfuric acid in the pretreatment solution to be 3 wt%, reacting in a sterilization pot at 130 ℃ for 30min, cooling, adjusting the pH to be 4, adding cellulase (the mass ratio of the cellulase to the pretreatment solution is 0.05: 100), reacting at 35 ℃ and the rotating speed of 120rpm for 10h, and centrifuging to obtain a solid containing protein and an enzymolysis solution containing monosaccharide.
Example 8
Protein extraction was performed according to the method of example 3, except that the step (4) was: and (3) mixing 8g of the solid containing the protein obtained in the step (3) with 20g of water, uniformly mixing with 80g of ethanol and 5g of sodium hydroxide, carrying out ultrasonic treatment at the temperature of 70 ℃ for 20min, filtering to obtain a mixed clear liquid, cooling the mixed clear liquid, adjusting the pH value to 5, centrifuging to collect a crude protein solid, and carrying out reduced pressure concentration and freeze drying on the crude protein solid to obtain protein powder.
Comparative example 1
Protein extraction was performed according to the method of example 3, except that the step (4) was: and (3) mixing 10g of the solid containing the protein obtained in the step (3) with 40g of water, uniformly mixing with 50g of ethanol and 3g of sodium hydroxide, carrying out ultrasonic treatment at the temperature of 70 ℃ for 20min, filtering to obtain a mixed clear liquid, and carrying out reduced pressure concentration and freeze drying on the mixed clear liquid to obtain protein powder.
Comparative example 2
Protein extraction was performed according to the method of example 3, except that the step (3) was: and (3) mixing 30g of the vinasse obtained in the step (2) with 300g of water to obtain a mixture A, mixing the mixture A with cellulase (the mass ratio of the cellulase to the mixture A is 0.05: 100), reacting for 8 hours at the temperature of 55 ℃ and the rotating speed of 300rpm, and centrifuging to obtain a solid containing protein and an enzymolysis liquid containing monosaccharide.
Comparative example 3
(1) Mixing 1000g of corn starch in dry weight with a certain amount of water until the dry matter concentration is 40%, adding 0.5g of alpha-amylase and adjusting the pH value to 4.3-4.5, liquefying at 95 ℃ for 2h to obtain a corn liquefied solution, adjusting the pH value of the corn liquefied solution to 4.3-4.5, adding 0.1g of saccharifying enzyme and 3g of ammonium nitrate, and inoculating with an amount of 5 × 108Inoculating the cfu/mL inoculum size into saccharomyces cerevisiae, and fermenting for 85h under the conditions that the temperature is 25 ℃ and the rotating speed is 150rpm to obtain fermentation liquor;
(2) distilling the ethanol fermentation liquor to remove ethanol to obtain fermented mash, filtering the fermented mash to obtain wet cake and fermented clear liquid, concentrating a part of the fermented clear liquid to obtain fermented thick liquid, mixing the fermented thick liquid with the wet cake, and freeze-drying to obtain vinasse;
(3) and (3) taking 30g of the vinasse obtained in the step (2), adding 100g of ethanol-water solution with volume concentration of 80%, performing ultrasonic treatment for 80min, centrifuging to obtain residual vinasse solid, mixing the residual vinasse solid with 300g of ethanol-water solution with volume concentration of 95% and 1200g of sodium hydroxide-water solution with mass concentration of 2%, performing centrifugation after extraction for 80min to obtain supernatant containing protein, adjusting the pH of the supernatant to 4.7, centrifuging, taking precipitate, and performing freeze drying to obtain protein powder.
Test example
The protein powders obtained in examples 1 to 8 and comparative examples 1 to 3 were used, and the protein content of the protein powders was measured, and the results are shown in Table 1; taking the enzymolysis liquid fermentation liquid prepared in the step (4) of the embodiment 1-the embodiment 8 and the comparative example 1 and the comparative example 2, measuring the concentration of ethanol in the enzymolysis liquid fermentation liquid, namely the content of ethanol in each liter of the enzymolysis liquid fermentation liquid, and the result is shown in the table 1.
As can be seen from the results in table 1, in examples 1 to 8, by using the method for extracting protein from distiller's grains provided by the present invention, compared with the methods in comparative examples 1 to 3, the protein powder obtained has high protein content and high extraction efficiency of protein from distiller's grains, and in addition, the enzymatic hydrolysate containing monosaccharide in the protein extraction process can be directly used as a culture medium for ethanol fermentation, so that the cost of the ethanol process can be effectively saved, and the economy of the whole ethanol process can be improved.
TABLE 1
The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.
Claims (10)
1. A method for extracting protein from vinasse is characterized by comprising the following steps:
(1) carrying out acid pretreatment, enzymolysis and solid-liquid separation I on the vinasse to obtain solid containing protein and enzymolysis liquid containing monosaccharide;
(2) mixing and extracting the solid containing the protein with organic alcohol and strong base, performing solid-liquid separation II to obtain mixed clear liquid, adjusting the pH of the mixed clear liquid to acidity, performing solid-liquid separation III to obtain crude protein solid, and drying the crude protein solid I to obtain the protein.
2. The method according to claim 1, wherein in the step (1), the preparation method of the vinasse comprises the following steps: separating ethanol from ethanol fermentation liquor to obtain fermented mash, performing solid-liquid separation IV on the fermented mash to obtain wet cake and fermented clear liquid, concentrating the fermented clear liquid to obtain fermented thick slurry, mixing the fermented thick slurry and the wet cake, and drying II.
3. The method of claim 2, wherein the ethanol fermentation broth is prepared by a process comprising: mixing a starchy raw material, alpha-amylase and a liquefying solvent for liquefaction, and then mixing the mixture with saccharifying enzyme, a nitrogen source and ethanol zymocyte I for fermentation I;
wherein the nitrogen source is selected from one or more of urea, ammonium sulfate and ammonium nitrate, and the ethanol fermentation bacteria I is selected from one or more of yeast, zymomonas mobilis, aspergillus and rhizopus.
4. The method according to claim 3, wherein the amount of the α -amylase is 0.01 to 0.05g, the amount of the saccharifying enzyme is 0.01 to 0.05g, the amount of the nitrogen source is 0.05 to 0.3g, and the amount of the liquefying solvent is 150-300g, relative to 100g of the starchy raw material by dry weight; the inoculation amount of the ethanol zymocyte I is 1 multiplied by 108-5×108cfu/mL；
Preferably, the condition for liquefaction is at least satisfied: the pH is 3.5-4.5, the temperature is 75-95 ℃, and the time is 2-5 h;
the condition of fermentation I at least satisfies: the temperature is 25-35 ℃, the rotation speed is 150-.
5. The method according to any one of claims 1 to 4, wherein in the step (1), the acid pretreatment comprises: mixing the vinasse with water, mixing the vinasse with an acid solution to obtain a pretreatment solution, and performing acid treatment on the pretreatment solution;
preferably, the mass ratio of the vinasse to the water is 1: 1-19;
the acid solution is selected from one or more of sulfuric acid solution, hydrochloric acid solution and nitric acid solution, and the concentration of the acid in the pretreatment solution is 0.5-3 wt%;
the acid treatment conditions at least satisfy: the temperature is 100 ℃ and 130 ℃, and the time is 30-90 min.
6. The method of claim 5, wherein in step (1), the enzymatic hydrolysis comprises: mixing the pretreatment solution after acid treatment with enzyme and then reacting;
preferably, the enzyme is selected from one or more of saccharifying enzyme, cellulase and xylanase, and the mass ratio of the enzyme I to the pretreatment solution after acid treatment is 0.05-1.4: 100, respectively;
the reaction conditions at least satisfy: the pH value is 4-5.5, the temperature is 45-55 ℃, the rotation speed is 200-300rpm, and the time is 30-64 h.
7. The method according to any one of claims 1 to 4, wherein in step (2), the amount of the organic alcohol is less than or equal to 90g, and the amount of the strong base is less than or equal to 5g, relative to 10g of the protein-containing solid on a dry weight basis;
the process of mixed extraction comprises the following steps: mixing the solid containing the protein with organic alcohol and strong base, and then carrying out ultrasonic treatment, wherein the ultrasonic treatment at least meets the following conditions: the temperature is 20-70 deg.C, and the time is 20-120 min.
8. The method according to any one of claims 1 to 4, wherein in the step (2), the pH of the mixed clear solution is adjusted to 4 to 5.
9. The method according to any one of claims 1 to 4, characterized in that the method further comprises: and (2) mixing the monosaccharide-containing enzymatic hydrolysate obtained in the step (1) with ethanol fermentation bacteria II to carry out fermentation II.
10. The method according to claim 9, wherein the ethanol fermentation bacteria II are selected from one or more of yeast, zymomonas mobilis, aspergillus and rhizopus;
the conditions of fermentation II at least satisfy: the temperature is 25-35 ℃, the rotating speed is 150-8-5×108cfu/mL。
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