CN104419734B - A kind of method using Immobilized yeast production ethyl alcohol - Google Patents

A kind of method using Immobilized yeast production ethyl alcohol Download PDF

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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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yeast
fermentation
ethyl alcohol
cassava
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CN104419734A (en
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胡世洋
刘海军
岳军
徐友海
朱庆伟
吕继萍
惠继星
王继艳
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Petrochina Co Ltd
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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
    • 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
    • C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
    • 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
    • C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
    • 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
    • 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

Abstract

The present invention relates to a kind of methods using Immobilized yeast production ethyl alcohol；It first by fresh cassava and cassava dry grinding, is mixed with water, adds in alpha amylase, obtain liquefier, heating sterilizing；Cool down saccharification again, adds carbohydrase and obtains fermentable sugars liquid, then cools down and ferment；Appropriate nitrogen source and nutritional ingredient are added during fermentation；Angel AADY is put into the activated yeast culture medium after sterilizing, is placed in shaken cultivation in water bath with thermostatic control；When cell number reaches 3.0 × 108A/ml~6.0 × 108Terminate to activate during a/ml；Yeast bacteria suspension is taken to be added in sodium alginate and polyethylene glycol oxide mixed solution, is added drop-wise in calcium chloride solution, forms bead, obtained gel, fixed yeast Multiplying culture；Fermentation, obtains the thick mash containing ethyl alcohol；This method material is food-grade, to nonhazardous of fermenting, can prepare feed in recycling afterwards of ferment, the mass concentration of ethyl alcohol reaches 13.6%~14.8% in mash, and alcohol fermentation is efficient.

Description

A kind of method using Immobilized yeast production ethyl alcohol
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)

  1. 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) ferment
    Fermentation 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. 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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CN106954880A (en) * 2016-10-13 2017-07-18 湖北中烟工业有限责任公司 A kind of method that immobilized yeast prepares cigarette apple extract
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