CN104419734B - Method for producing ethanol by fermentation of immobilized yeast - Google Patents
Method for producing ethanol by fermentation of immobilized yeast Download PDFInfo
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- CN104419734B CN104419734B CN201310397826.1A CN201310397826A CN104419734B CN 104419734 B CN104419734 B CN 104419734B CN 201310397826 A CN201310397826 A CN 201310397826A CN 104419734 B CN104419734 B CN 104419734B
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 title claims abstract description 65
- 238000000855 fermentation Methods 0.000 title claims abstract description 46
- 230000004151 fermentation Effects 0.000 title claims abstract description 46
- 240000004808 Saccharomyces cerevisiae Species 0.000 title claims abstract description 45
- 238000004519 manufacturing process Methods 0.000 title claims abstract description 16
- 240000003183 Manihot esculenta Species 0.000 claims abstract description 68
- 235000016735 Manihot esculenta subsp esculenta Nutrition 0.000 claims abstract description 68
- 238000000034 method Methods 0.000 claims abstract description 21
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 20
- 230000001954 sterilising effect Effects 0.000 claims abstract description 17
- 238000002156 mixing Methods 0.000 claims abstract description 14
- 239000007788 liquid Substances 0.000 claims abstract description 13
- 235000010413 sodium alginate Nutrition 0.000 claims abstract description 12
- 239000000725 suspension Substances 0.000 claims abstract description 10
- 230000004913 activation Effects 0.000 claims abstract description 9
- 102000004139 alpha-Amylases Human genes 0.000 claims abstract description 9
- 108090000637 alpha-Amylases Proteins 0.000 claims abstract description 9
- 229940024171 alpha-amylase Drugs 0.000 claims abstract description 9
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims abstract description 8
- 239000000661 sodium alginate Substances 0.000 claims abstract description 8
- 229940005550 sodium alginate Drugs 0.000 claims abstract description 8
- 235000000346 sugar Nutrition 0.000 claims abstract description 8
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims abstract description 6
- 239000001963 growth medium Substances 0.000 claims abstract description 6
- 239000011259 mixed solution Substances 0.000 claims abstract description 6
- 238000004659 sterilization and disinfection Methods 0.000 claims abstract description 5
- 235000019441 ethanol Nutrition 0.000 claims description 29
- 239000002994 raw material Substances 0.000 claims description 20
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 10
- 239000002202 Polyethylene glycol Substances 0.000 claims description 10
- 229920001223 polyethylene glycol Polymers 0.000 claims description 10
- 238000002360 preparation method Methods 0.000 claims description 10
- 229920002472 Starch Polymers 0.000 claims description 9
- 238000005516 engineering process Methods 0.000 claims description 9
- 238000000386 microscopy Methods 0.000 claims description 9
- 238000004321 preservation Methods 0.000 claims description 9
- 235000019698 starch Nutrition 0.000 claims description 9
- 239000008107 starch Substances 0.000 claims description 9
- 238000003756 stirring Methods 0.000 claims description 9
- 108010089934 carbohydrase Proteins 0.000 claims description 8
- 239000002002 slurry Substances 0.000 claims description 8
- 239000004576 sand Substances 0.000 claims description 7
- 241000894006 Bacteria Species 0.000 claims description 6
- NBIIXXVUZAFLBC-UHFFFAOYSA-L Phosphate ion(2-) Chemical compound OP([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-L 0.000 claims description 6
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims description 6
- 239000000654 additive Substances 0.000 claims description 6
- 230000000996 additive effect Effects 0.000 claims description 6
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 6
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 6
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 6
- 239000011591 potassium Substances 0.000 claims description 6
- 229910052700 potassium Inorganic materials 0.000 claims description 6
- 238000010792 warming Methods 0.000 claims description 6
- 238000013019 agitation Methods 0.000 claims description 5
- 238000004140 cleaning Methods 0.000 claims description 5
- 238000009837 dry grinding Methods 0.000 claims description 5
- 230000000694 effects Effects 0.000 claims description 5
- 239000004615 ingredient Substances 0.000 claims description 5
- 229910052757 nitrogen Inorganic materials 0.000 claims description 5
- 235000016709 nutrition Nutrition 0.000 claims description 5
- 150000008163 sugars Chemical class 0.000 claims description 5
- 239000003643 water by type Substances 0.000 claims description 5
- 239000011324 bead Substances 0.000 claims description 4
- 238000005406 washing Methods 0.000 claims description 4
- 230000008859 change Effects 0.000 claims description 3
- 239000001110 calcium chloride Substances 0.000 claims description 2
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 2
- 239000000243 solution Substances 0.000 claims description 2
- 239000000843 powder Substances 0.000 claims 2
- ZGBSOTLWHZQNLH-UHFFFAOYSA-N [Mg].S(O)(O)(=O)=O Chemical compound [Mg].S(O)(O)(=O)=O ZGBSOTLWHZQNLH-UHFFFAOYSA-N 0.000 claims 1
- 230000010355 oscillation Effects 0.000 claims 1
- 230000035755 proliferation Effects 0.000 claims 1
- 239000000463 material Substances 0.000 abstract description 5
- 108090000790 Enzymes Proteins 0.000 abstract description 2
- 102000004190 Enzymes Human genes 0.000 abstract description 2
- 229920003171 Poly (ethylene oxide) Polymers 0.000 abstract description 2
- 229940088598 enzyme Drugs 0.000 abstract description 2
- 238000010438 heat treatment Methods 0.000 abstract description 2
- -1 polyoxyethylene Polymers 0.000 abstract description 2
- 238000001816 cooling Methods 0.000 abstract 2
- 230000003213 activating effect Effects 0.000 abstract 1
- 238000012258 culturing Methods 0.000 abstract 1
- 235000013305 food Nutrition 0.000 abstract 1
- 231100000252 nontoxic Toxicity 0.000 abstract 1
- 230000003000 nontoxic effect Effects 0.000 abstract 1
- 235000015097 nutrients Nutrition 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 15
- 239000003153 chemical reaction reagent Substances 0.000 description 8
- 210000005253 yeast cell Anatomy 0.000 description 7
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 6
- 239000012530 fluid Substances 0.000 description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 3
- 239000001888 Peptone Substances 0.000 description 3
- 108010080698 Peptones Proteins 0.000 description 3
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 3
- 239000005864 Sulphur Substances 0.000 description 3
- 229940041514 candida albicans extract Drugs 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 239000011777 magnesium Substances 0.000 description 3
- 229910052749 magnesium Inorganic materials 0.000 description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 3
- 235000019341 magnesium sulphate Nutrition 0.000 description 3
- 235000019319 peptone Nutrition 0.000 description 3
- 235000013599 spices Nutrition 0.000 description 3
- 239000012138 yeast extract Substances 0.000 description 3
- 241000196324 Embryophyta Species 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 238000000227 grinding Methods 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 239000004382 Amylase Substances 0.000 description 1
- 108010065511 Amylases Proteins 0.000 description 1
- 102000013142 Amylases Human genes 0.000 description 1
- 239000004372 Polyvinyl alcohol Substances 0.000 description 1
- 150000001336 alkenes Chemical class 0.000 description 1
- 235000019418 amylase Nutrition 0.000 description 1
- 235000010410 calcium alginate Nutrition 0.000 description 1
- 239000000648 calcium alginate Substances 0.000 description 1
- 229960002681 calcium alginate Drugs 0.000 description 1
- OKHHGHGGPDJQHR-YMOPUZKJSA-L calcium;(2s,3s,4s,5s,6r)-6-[(2r,3s,4r,5s,6r)-2-carboxy-6-[(2r,3s,4r,5s,6r)-2-carboxylato-4,5,6-trihydroxyoxan-3-yl]oxy-4,5-dihydroxyoxan-3-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylate Chemical compound [Ca+2].O[C@@H]1[C@H](O)[C@H](O)O[C@@H](C([O-])=O)[C@H]1O[C@H]1[C@@H](O)[C@@H](O)[C@H](O[C@H]2[C@H]([C@@H](O)[C@H](O)[C@H](O2)C([O-])=O)O)[C@H](C(O)=O)O1 OKHHGHGGPDJQHR-YMOPUZKJSA-L 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000011049 filling Methods 0.000 description 1
- 239000012847 fine chemical Substances 0.000 description 1
- 210000001822 immobilized cell Anatomy 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000009434 installation Methods 0.000 description 1
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 229920002451 polyvinyl alcohol Polymers 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 230000002000 scavenging effect Effects 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
Classifications
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- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
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- 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
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- 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
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Abstract
A process for preparing alcohol by fermenting immobilized yeast includes such steps as crushing fresh cassava and cassava, mixing with water, adding α -amylase to obtain liquefied liquid, heating for sterilization, cooling for saccharification, adding saccharifying enzyme to obtain fermentable sugar liquid, cooling for fermentation, adding N source and nutrients, adding Angel wine-brewing active dry yeast to sterilized yeast activating culture medium, vibration culturing in constant-temp water bath until the number of cells reaches 3.0 × 10
8one/ml-6.0X 10
8Ending activation at one/ml; adding the yeast suspension into a mixed solution of sodium alginate and polyoxyethylene, dropwise adding the mixed solution into a calcium chloride solution to form small balls, obtaining gel, and carrying out immobilized yeast propagation culture; fermenting to obtain thick mash containing ethanol; the material of the method is food grade, is non-toxic to fermentation, and can be recycled to prepare the feed after fermentation is finished, the mass concentration of ethanol in mash reaches 13.6-14.8%, and the ethanol fermentation efficiency is high.
Description
Technical field:
The invention belongs to Fermentation Engineerings and biology, engineering in medicine field, are more particularly to produced using Immobilized yeast
The method of ethyl alcohol.
Background technology:
Cassava is a kind of perennial plant, originates in the hilly country of subtropical and tropical zones, it has the processing performance good
And the advantages of not striving with grain ground, and with it is thick it is raw easily plant, drought-enduring, resistance to lean, the adaptable, characteristics such as per unit area yield is high, it is considered to be most
One of economic energy crop.
It is typically all that fresh cassava is used to produce ethyl alcohol as raw material at present, existing main problem is the wine of fermenting-ripening wine with dregs
Part low, yeast cells density is low in filtration difficulty, zymotic fluid, inhibitory action, sugar solution speed of the ethyl alcohol to yeast cells in zymotic fluid
Rate is low, fermentation rate is low and utilization rate of equipment and installations, labor productivity, energy utilization rate are all relatively low, and production cost is higher.It is improved
Method is to improve fermentation liquid concentration and barm cell concentration.
Patent CN101629189A is that the fresh cassava after first time is crushed is sized mixing, and hot water is used in heating liquefaction filtering
Wash filter residue, recycle filter residue in residual sugar, the filtrate being obtained by filtration can be used for first time fresh cassava crushing, by the use of first time filtrate as
The crushing liquid of second of fresh cassava, the slurry crushed are used for performing thick mash alcohol fermentation as raw material.But the method energy consumption water consume
It is excessively high.Patent CN101245354A is that raw material uses two-stage spice slurrying production technology using dry cassava, and level-one spice slurrying obtains
The relatively low dilute slurry of viscosity carries out settling operation of removing sand, and thin pulp obtains denseer slurry using secondary tapioca starch spice slurrying.
But the raw material of the method rate of removing sand is low, is unfavorable for later stage fermentation.
But the above method all can not be avoided fundamentally in alcohol fermentation, yeast cells density is low in zymotic fluid, sends out
Ethyl alcohol is to the inhibitory action of yeast cells in zymotic fluid, with the rising of ethanol product concentration in zymotic fluid, when ethyl alcohol mass concentration
When reaching more than 9%, yeast cells will stop growing completely, and fermentation can stay cool.So individually improve fermentation liquid
Concentration is unfavorable for improving fermentation rate, can not reduce alcohol production cost.
The immobilization technology of yeast cells is a kind of new and high technology for producing ethyl alcohol.Yeast in zymotic fluid can not only be solved
The problem of cell quantity, can also improve inhibition of the yeast cells to ethyl alcohol.But it is raw that immobilization technology is applied to industry
In production, a series of problems, such as first having to solve fixation support softening in production, rupture, cost and usage time, at present
Yeast immobilization is largely using investments such as calcium alginate, polyvinyl alcohol, but there are cost is higher, mechanical strength is low, fixed
The shortcomings of change process is cumbersome, carrier ruckbildung.
The content of the invention:
The object of the present invention is to provide a kind of methods that Immobilized Yeast ethyl alcohol is utilized using cassava as raw material.It can either
The Cassava-based ethanol production method of barm cell concentration can be improved again by improving fermentation liquid concentration.
A kind of method that Immobilized Yeast ethyl alcohol is utilized using cassava as raw material of the present invention, the step of preparation and
Condition is as follows:
Fresh cassava and Tapioca chips is used to produce ethyl alcohol using Immobilized yeast for raw material.It is raw materials used have fresh cassava and
Tapioca chips, alpha-amylase, carbohydrase, sodium alginate, polyethylene glycol oxide, Angel highly active dry yeast.
The present invention is divided into three steps, is the preparation process of fermentation liquid first, followed by yeast fixes the preparation of cell
Process is finally fermented.
In the preparation process of fermentation liquid, raw material grinding particle size and its mixed proportion are controlled(Fresh cassava granularity is in 1.4mm
~1.7mm, Tapioca chips granularity is in 0.21mm~0.25mm.It is 1 ︰, 2 ︰ 5.5 by the weight ratio of fresh cassava Fen ︰ Tapioca chips Fen ︰ water
~8 mixing), control the additive amount and temperature of alpha-amylase and carbohydrase:Alpha-amylase dosage is 10U/g~20U/g, is liquefied
Temperature keeps the temperature 120 minutes~140 minutes at 75 DEG C~95 DEG C, is warming up to 105 DEG C~110 DEG C afterwards and sterilizes 5 minutes~10 points
Clock.Be saccharified enzyme dosage 240U/g~260U/g, and saccharification temperature keeps the temperature 20 minutes~60 minutes, reach raw material at 56 DEG C~62 DEG C
To the purpose being fully saccharified.
The preparation process of cell is fixed in yeast, first carries out activated yeast, cell number reaches 3.0 × 108A/mL~6.0
×108Terminate to activate during a/mL, then prepare immobilized cell, sodium alginate and polyoxyethylene are controlled in preparation process
Alkene mixed proportion, sodium alginate viscosity is in 1.05PaS~1.15PaS, and polyethylene glycol oxide molecular weight is more than 300,000.2%~
3% sodium alginate and 0.1%~0.5% polyethylene glycol oxide are prepared by mixing into gel.So be conducive to improve the intensity for curing cell.
During the fermentation major control well fix cell filling rate be conducive to ferment between 45%~55%.
The present invention produces ethyl alcohol using mixed raw material, the weight ratio of fresh cassava Fen ︰ Tapioca chips Fen ︰ water in 1 ︰, 2 ︰ 5.5~8,
Grin-ding energ7 is so reduced, it is cost-effective.
For the immobilization material of the present invention using sodium alginate, polyethylene glycol oxide, material price is cheap and belongs to food-grade
Raw material recycling can prepare feed to no toxic action of fermenting after fermentation.Using its fermentation of immobilization fermentation technique
Between the mass concentration of mash end ethyl alcohol can reach 13.6%~14.8%, alcohol fermentation efficiency is substantially increased.
Specific embodiment:
Material and reagent:
Fresh cassava:Place of production Beihai Fisheries Base Guangxi Province, content of starch 28%, moisture 48%;
Tapioca chips:Place of production Beihai Fisheries Base Guangxi Province, content of starch 67%, moisture 12%;
Super highly active dry yeast:Angel Yeast Co., Ltd produces;
Heatproof a- amylase (2000U/g):Tianjin good fortune morning chemical reagent factory produces;
Carbohydrase(100000U/g):The extensive and profound in meaning star biotechnology Co., Ltd production in Beijing;
Sodium alginate (1.05PaS-1.15PaS):Tianjin good fortune morning chemical reagent factory produces, experiment reagent;
Polyethylene glycol oxide(300000 molecular weight):Tianjin Feng Yue Chemical Companies produce, and analysis is pure;
Calcium chloride:Tianjin day is produced up to scavenging material Fine Chemical Works, and analysis is pure;
Magnesium sulfate:The production of Tianjin recovery development in science and technology Co., Ltd, analysis are pure;
Ammonium sulfate:Tianjin Rui Jin spy Chemical Company produces, and analysis is pure;
Potassium dihydrogen phosphate:Tianjin good fortune morning chemical reagent factory produces, and analysis is pure;
Yeast extract:The extensive and profound in meaning star biotechnology Co., Ltd production in Beijing, biochemical reagents;
Glucose:Tianjin Chemical Reagents Factory No.1 produces, and analysis is pure;
Peptone:Tianjin Ying Bo biochemical reagents Co., Ltd, biochemical reagents:
YPD activation mediums:Yeast extract 10g/L, peptone 20g/L, glucose 20g/L.
Proliferated culture medium:Glucose 5g/L, peptone 0.5g/L, yeast extract 0.5g/L, magnesium sulfate 0.1g/L, biphosphate
Potassium 0.1g/L.
Comparative example 1:
1)Using tapioca starch zymotechnique
1. Tapioca chips is carried out cleaning and processing of removing sand first, the crushing of raw material is then carried out.Cassava dry grinding makes its grain
Degree is in 0.21mm or so, 70 sieve meshes.
2. the tapioca starch after above-mentioned 1. crushing is mixed with water, it is mixed for 1 ︰ 2 by the weight ratio of Tapioca chips Fen ︰ water
It closes, total fermentation volume 3L, then heats to 65 DEG C, stirring is sized mixing 40 minutes, achievees the purpose that be completely dissolved.
3. it is 10U/g that alpha-amylase dosage is added in slurries 2., temperature control keeps the temperature 120 points in 75 DEG C, pH value 4.8
Clock obtains liquefier, is warming up to 105 DEG C afterwards and sterilizes 5 minutes, achievees the effect that gelatinization completely and sterilizing.56 DEG C are cooled to again to reach
To saccharification temperature, pH value is adjusted 3.8, adds 240U/g carbohydrase, heat preservation obtains fermentable sugars liquid in 20 minutes, then cools to
30 DEG C ferment.Appropriate nitrogen source and nutritional ingredient are added during fermentation, ammonium sulfate additive amount exists in 0.3g/L, magnesium sulfate dosage
0.05g/L, biphosphate potassium application rate are in 0.15g/L.
2)The activation of dry ferment:
1. take 4g Angel highly active dry yeasts be put into sterilizing after 200m1 YPD activated yeast culture mediums in, be placed in
Shaken cultivation 30 minutes in 30 DEG C of waters bath with thermostatic control.Sterilised yeast suspension microscopy is taken, when cell number reaches 3.0 × 108During a/ml or so,
Terminate activation.
3)Fermentation:
Fermentation temperature is at 30 DEG C, agitation revolution 150rpm/min, when fermentation time 48 is small, ethyl alcohol in the thick mash that ferments eventually
Mass concentration reaches 11.6%.
Embodiment 1:
Invent a kind of method that Immobilized Yeast ethyl alcohol is utilized using cassava as raw material.
1)Using fresh cassava and Tapioca chips mixed fermentation technology
1. fresh cassava and Tapioca chips are carried out cleaning and processing of removing sand first, the crushing of raw material is then carried out.Fresh cassava is adopted
With comminuting method fine crushing, make its granularity in 1.4mm or so, 12 sieve meshes.Again by cassava dry grinding, make its granularity in 0.21mm or so,
70 sieve meshes
2. the tapioca starch after above-mentioned 1. crushing is mixed with water, by the weight ratio of fresh cassava Fen ︰ Tapioca chips Fen ︰ water
It is mixed for 1 ︰, 2 ︰ 5.5, then heats to 95 DEG C of stirrings and size mixing 30 minutes, achieve the purpose that be completely dissolved.
3. it is 10U/g that alpha-amylase dosage is added in slurries 2., temperature control is at 75 DEG C, between pH value is 4.8, heat preservation
It obtains liquefier within 120 minutes, is warming up to 105 DEG C afterwards and sterilizes 5 minutes, achieve the effect that gelatinization completely and sterilizing.It cools to again
56 DEG C reach saccharification temperature, adjust pH value between 3.8, add 240U/g carbohydrase, and heat preservation obtains fermentable sugars in 20 minutes
Liquid, then cool to 30 DEG C and ferment.Appropriate nitrogen source and nutritional ingredient are added during fermentation, ammonium sulfate additive amount is in 0.3g/L, sulphur
Sour magnesium dosage is in 0.05g/L, biphosphate potassium application rate in 0.15g/L.
2)The activation and immobilization of dry ferment:
1. take 4g Angel highly active dry yeasts be put into sterilizing after 200m1 YPD activated yeast culture mediums in, be placed in
Shaken cultivation 140 minutes in 30 DEG C of waters bath with thermostatic control.Sterilised yeast suspension microscopy is taken, when cell number reaches 6.0 × 108During a/ml or so
Terminate activation.
2. aseptically, take 1. yeast bacteria suspension be added to 2% sodium alginates of the 100m1 Jing Guo sterilization treatment and
It in 0.5% polyethylene glycol oxide mixed solution, is added drop-wise to dropwise in 2% calcium chloride solution with syringe after mixing, forms grain
It stirs per half an hour and once stands overnight in the bead of 3mm, balanced reaction 60 minutes at 3 DEG C in footpath.Obtained gel sterilizing
Water washing 4 times is fixed yeast Multiplying culture afterwards, the gel of preparation is placed in proliferated culture medium that constant temperature shakes at 30 DEG C
Swing Multiplying culture 30 minutes, microscopy free cell number is up to 108A/more than ml.
3)Fermentation:
Fermentation temperature is at 30 DEG C, and gel-filled rate is in 45%, agitation revolution 100rpm/min, when fermentation time 48 is small, whole hair
The mass concentration of ethyl alcohol reaches 13.6% in ferment thick mash.
Embodiment 2:
Invent a kind of method that Immobilized Yeast ethyl alcohol is utilized using cassava as raw material.
1)Using fresh cassava and Tapioca chips mixed fermentation technology
1. fresh cassava and Tapioca chips are carried out cleaning and processing of removing sand first, the crushing of raw material is then carried out.Fresh cassava is adopted
With comminuting method fine crushing, make its granularity in 1.7mm or so, 10 sieve meshes.Again by cassava dry grinding, make its granularity in 0.25mm or so,
60 sieve meshes.
2. the tapioca starch after above-mentioned 1. crushing is mixed with water, by the weight ratio of fresh cassava Fen ︰ Tapioca chips Fen ︰ water
It is mixed for 1 ︰, 2 ︰ 8, then heats to 55 DEG C of stirrings and size mixing 60 minutes, achieve the purpose that be completely dissolved.
3. it is 20U/g that alpha-amylase dosage is added in slurries 2., temperature control is at 95 DEG C, between pH value is 6.2, heat preservation
It obtains liquefier within 140 minutes, is warming up to 110 DEG C afterwards and sterilizes 10 minutes, achieve the effect that gelatinization completely and sterilizing.It cools to again
62 DEG C reach saccharification temperature, adjust pH value between 4.6, add 260U/g carbohydrase, and heat preservation obtains fermentable sugars in 60 minutes
Liquid, then cool to 36 DEG C and ferment.Appropriate nitrogen source and nutritional ingredient are added during fermentation, ammonium sulfate additive amount is in 0.5g/L, sulphur
Sour magnesium dosage is in 0.10g/L, biphosphate potassium application rate in 0.20g/L.
2)The activation and immobilization of dry ferment:
1. take 8g Angel highly active dry yeasts be put into sterilizing after 400m1 YPD activated yeast culture mediums in, be placed in
Shaken cultivation 60 minutes in 36 DEG C of waters bath with thermostatic control.Sterilised yeast suspension microscopy is taken, when cell number reaches 3.0 × 108Terminate to live during a/ml
Change.
2. aseptically, take 1. yeast bacteria suspension be added to 3% sodium alginates of the 100m1 Jing Guo sterilization treatment and
It in 0.1% polyethylene glycol oxide mixed solution, is added drop-wise to dropwise in 5% calcium chloride solution with syringe after mixing, forms grain
Footpath is in the bead of 5mm, and balanced reaction 180 minutes at 5 DEG C, per half an hour, stirring is once stood overnight.Obtained gel is with going out
Bacterium water washing 2 times is fixed yeast Multiplying culture afterwards, the gel of preparation is placed in proliferated culture medium, constant temperature at 36 DEG C
Vibrate Multiplying culture 120 minutes, microscopy free cell number is up to 108A/more than ml.
3)Fermentation:
Fermentation temperature is at 36 DEG C, and gel-filled rate is in 55%, agitation revolution 200rpm/min, when fermentation time 75 is small, whole hair
The mass concentration of ethyl alcohol reaches 14.8% in ferment thick mash.
Embodiment 3:
Invent a kind of method that Immobilized Yeast ethyl alcohol is utilized using cassava as raw material.
1)Using fresh cassava and Tapioca chips mixed fermentation technology
1. fresh cassava and Tapioca chips are carried out cleaning and processing of removing sand first, the crushing of raw material is then carried out.Fresh cassava is adopted
With comminuting method fine crushing, making its granularity, 10 sieve meshes are between 12 sieve meshes in 1.5mm or so.Again by cassava dry grinding, its granularity is made to exist
0.23mm or so, 65 sieve meshes.
2. the tapioca starch after above-mentioned 1. crushing is mixed with water, by the weight ratio of fresh cassava Fen ︰ Tapioca chips Fen ︰ water
It is mixed for 1 ︰, 2 ︰ 6, then heats to 80 DEG C of stirrings and size mixing 40 minutes, achieve the purpose that be completely dissolved.
3. it is 15U/g that alpha-amylase dosage is added in slurries 2., temperature control is at 80 DEG C, between pH value is 5.2, heat preservation
It obtains liquefier within 130 minutes, is warming up to 105 DEG C afterwards and sterilizes 8 minutes, achieve the effect that gelatinization completely and sterilizing.It cools to again
58 DEG C reach saccharification temperature, adjust pH value between 4.2, add 250U/g carbohydrase, and heat preservation obtains fermentable sugars in 40 minutes
Liquid, then cool to 32 DEG C and ferment.Appropriate nitrogen source and nutritional ingredient are added during fermentation, ammonium sulfate additive amount is in 0.4g/L, sulphur
Sour magnesium dosage is in 0.08g/L, biphosphate potassium application rate in 0.18g/L.
2)The activation and immobilization of dry ferment:
1. take 6g Angel highly active dry yeasts be put into sterilizing after 300m1 YPD activated yeast culture mediums in, be placed in
Shaken cultivation 80 minutes in 32 DEG C of waters bath with thermostatic control.Sterilised yeast suspension microscopy is taken, when cell number reaches 4.5 × 108A/ml or so Shi Jie
Beam activates.
2. aseptically, take 1. yeast bacteria suspension be added to 2.5% sodium alginates of the 100m1 Jing Guo sterilization treatment and
It in 0.25% polyethylene glycol oxide mixed solution, is added drop-wise to dropwise in 3% calcium chloride solution with syringe after mixing, forms grain
It stirs per half an hour and once stands overnight in the bead of 4mm, balanced reaction 120 minutes at 4 DEG C in footpath.Obtained gel is with going out
Bacterium water washing 3 times is fixed yeast Multiplying culture afterwards, the gel of preparation is placed in proliferated culture medium, constant temperature at 32 DEG C
Vibrate Multiplying culture 60 minutes, microscopy free cell number is up to 108A/more than ml.
3)Fermentation:
Fermentation temperature is at 32 DEG C, and gel-filled rate is in 50%, agitation revolution 150rpm/min, when fermentation time 60 is small, whole hair
The mass concentration of ethyl alcohol reaches 14.2% in ferment thick mash.
Claims (2)
- A kind of 1. method using Immobilized yeast production ethyl alcohol, it is characterised in that:1) fresh cassava and Tapioca chips mixed fermentation technology are used:1. fresh cassava and Tapioca chips are carried out cleaning and processing of removing sand first, the crushing of raw material is then carried out, fresh cassava is using thin Broken comminuting method makes its granularity spare between 1.4mm~1.7mm, then by cassava dry grinding, make its granularity 0.21mm~ Between 0.25mm;2. the tapioca starch after above-mentioned 1. crushing is mixed with water, by fresh cassava powder:Cassava dry powder:The weight ratio of water is 1: 2:5.5~8 mixing, then heat to 55 DEG C~110 DEG C stirrings and size mixing 30 minutes~60 minutes, reach and be completely dissolved;3. it is l0U/g~20U/g that alpha-amylase dosage is added in slurries 2., temperature control at 75 DEG C~95 DEG C, pH value for 4.8~ Between 6.2, heat preservation obtains liquefier in 120 minutes~140 minutes, is warming up to 105 DEG C~110 DEG C afterwards and sterilizes 5 minutes~10 points Clock achievees the effect that gelatinization completely and sterilizing;56 DEG C~62 DEG C are cooled to again and reaches saccharification temperature, adjust pH value 3.8~4.6 Between, 240U/g~260U/g carbohydrase is added, heat preservation obtains fermentable sugars liquid in 20 minutes~60 minutes, then cools to 30 DEG C ~36 DEG C ferment;Appropriate nitrogen source and nutritional ingredient are added during fermentation, ammonium sulfate additive amount is in 0.3g/L~0.5g/L, sulfuric acid Magnesium dosage is in 0.05g/L~0.10g/L, and biphosphate potassium application rate is in 0.15g/L~0.20g/L;2) activation and immobilization of dry ferment:1. take 4g~8g Angel AADYs be put into sterilizing after 200m1~400m1 YPD activated yeast culture mediums In, it is placed in shaken cultivation 60 minutes~140 minutes in 30 DEG C~36 DEG C waters bath with thermostatic control;Sterilised yeast suspension microscopy is taken, when cell number reaches To 3.0 × 108A/ml~6.0 × 108Terminate to activate during a/ml;2. aseptically, take 1. yeast bacteria suspension be added to 2%~3% sodium alginates of the 100m1 Jing Guo sterilization treatment and In 0.1%~0.5% polyethylene glycol oxide mixed solution, 2%~5% calcium chloride is added drop-wise to dropwise with syringe after mixing In solution, grain size is formed in the bead of 3mm~5mm, balanced reaction 60 minutes~180 minutes, the balance at 3 DEG C~5 DEG C Per half an hour, stirring once, is then stood overnight during reaction;Obtained gel uses sterilizing water washing 2 times~4 times, is fixed afterwards Change Yeast proliferation culture, the gel of preparation is placed in proliferated culture medium, constant temperature oscillation Multiplying culture 30 divides at 30 DEG C~36 DEG C Clock~120 minute, microscopy free cell number is up to 108A/more than ml;3) fermentFermentation temperature is at 30 DEG C~36 DEG C, and gel-filled rate is 45%~55%, agitation revolution 100rpm~200rpm, during fermentation Between 48 it is small when~75 it is small when, the mass concentration of ethyl alcohol reaches 13.6%~14.8% in the thick mash that ferments eventually.
- 2. the method according to claim 1 using Immobilized yeast production ethyl alcohol, it is characterised in that:Sodium alginate Viscosity is in 1.05PaS~1.15PaS, and polyethylene glycol oxide molecular weight is more than 300,000.
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