CN107858298A - One plant of thermotolerant ethanol fermentation yeast bacterial strain and its structure - Google Patents
One plant of thermotolerant ethanol fermentation yeast bacterial strain and its structure Download PDFInfo
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Abstract
The invention discloses the screening of one plant of thermotolerant ethanol fermentation yeast and construction method.Using strains A Y12 G as starting strain, ARTP plasma mutagenesis is carried out, primary dcreening operation obtains 150 plants of bacterium under the conditions of 37 DEG C, then carries out genome rearrangement to this 150 plants of bacterium, and primary dcreening operation obtains 137 plants of bacterium under the conditions of 37 DEG C.This 137 plants of bacterium are carried out with 35 DEG C of corn hydrolyzate fermentations, filter out 14 plants of bacterium, 14 plants of bacterium are subjected to 35 DEG C of simultaneous saccharification and fermentations and measure 48h cell survival rates, filter out the bacterial strain that 7 plant height temperature fermenting properties and 48h cell survival rates have been lifted, this 7 plants of bacterium are carried out with 41 DEG C and 42 DEG C of high temperature acclimation, the final plant height temperature fermenting property that obtains is excellent, the strain X 130 that 48h cell survival rates are obviously improved.Compared with starting strain AY12 G, X 130 improves 84.68% in 48h cell survival rate, and 35 DEG C of simultaneous saccharification and fermentation performances do not have significant change, and the addition acid protease after fermentation cycle is reduced 12h.
Description
Technical field：
The present invention relates to genetic engineering field, is specifically related to one plant of thermotolerant ethanol fermentation yeast bacterial strain and its construction method.
Background technology：
Alcohol fuel is the gasoline instead fuel the most ripe that the current whole world is generally acknowledged.As a kind of liquid that can be regenerated
Fuel, alcohol fuel are alleviating atmosphere pollution, are reducing greenhouse gas emission, reduction to Imported oil dependence, activation rural economy
And improve Household income etc. and all play a very important role, therefore promoted in the multiple countries and regions in the whole world
Use.Alcohol fuel has become one of regenerative resource paid close attention to the most in the world.The yield of global alcohol fuel in 2015
Reach 98,300,000,000 liters, account for total biological liquid fuel (including ethanol, biodiesel and hydrogenated vegetable oil) yield (about 130,700,000,000
Rise) 75%, turn into the most important liquid recyclable fuel in the whole world, and the gasoline instead fuel that field of traffic is generally acknowledged.20 generation
Record the seventies, some scholars are in the research process to cellulose fermentation producing and ethanol, in order to prevent the suppression of sugar accumulation and end-product
Make and use, and then improve the catalyzing hydrolysis efficiency of cellulase, it is proposed that simultaneous saccharification and fermentation (Simultaneous
Saccharification and Fermentation, SSF) pattern, and receive extensive attention.The dry method of main flow at present
Ethanol all employs simultaneous saccharification and fermentation technology, i.e., adds carbohydrase and yeast in fermentation tank simultaneously, and carbohydrase is by liquid
The dextrin that chemical industry section is obtained is further broken into the utilizable reduced sugar of yeast, and reduced sugar is converted into ethanol by yeast.So
And have in SSF a sixty-four dollar question be exactly optimal hydrolysis temperature (45 DEG C~50 DEG C) and ethanol fermentation temperature (25 DEG C~
35 DEG C) it is uncoordinated, this causes the fermentation of saccharomycete and the enzymolysis of cellulose is difficult to synchronous progress, therefore seed selection thermotolerant yeast bacterium
It is the key for solving this problem.
In the fermentation process of ethanol, when the temperature of fermentation liquid is more than 38 DEG C, it must just be dropped using cooling water
Temperature, otherwise suppression will be produced to the activity of saccharomyces cerevisiae, influence the yield of ethanol and the utilization rate of reduced sugar, increase production
Cost.Hot fermentation has that fermenting speed is fast, is more suitable for simultaneous saccharification and fermentation technique, shortens fermentation period, saves cooling water
Dosage, reduce fermentation raw material viscosity, reduce the advantages that production cost, therefore, the resistant to elevated temperatures Wine brewing yeast strain of seed selection is in industry
Application aspect is significant.This research is under saccharomyces cerevisiae AY12 genetic background, with reference to the mutagenesis of ARTP ions, genome
The breeding technique such as rearrangement and high temperature acclimation, seed selection thermotolerant ethanol fermentation yeast.But on thermotolerant ethanol fermentation yeast, do not have also at present
There is the marketable product of comparative maturity.
The content of the invention：
Present invention solves the technical problem that it is to construct one plant using the mutagenesis of ARTP ions, genome rearrangement and high temperature acclimation
Thermotolerant ethanol fermentation yeast bacterial strain.
In order to solve the above technical problems, the present invention adopts the following technical scheme that：
Wine brewing yeast strain provided by the invention is the yeast strain for having to high temperature certain tolerance.High tolerance is obtained to make
The yeast strain that sets out of brewer yeast bacterial strain is saccharomyces cerevisiae (Saccharomyces cerevisiae) CICC32315, is stored in
Chinese industrial Microbiological Culture Collection administrative center, the public are commercially available.
Described thermotolerant yeast heat-resisting ability compared with original strain under same growth conditions is doubled
Above and salt resistance ability is significantly stronger than original strain.
Ethanol production also significantly improves the bacterial strain filtered out compared with Angel high temperature resistant dry ferment after domestication, and
Residual sugar content has also declined.Cell survival rate also significantly improves simultaneously.
Because the history of life of saccharomyces cerevisiae has particularity, its monoploid and double somatocyte can be with the shapes of trophosome
Formula is present.The effect of mutagens processing saccharomyces cerevisiae is random, when handling haploid cell, it is possible to several genes occur
Undergo mutation simultaneously, but wherein some mutation can cause the dead situation of bacterial strain.Which results in screening process it is some just
To the loss of mutation；On the contrary, if mutagenesis is carried out with diploid cell, even if generating a lethal mutation, as long as its equipotential
The mutation does not occur for gene, then bacterial strain can still survive, so as to expand the scope of catastrophe point.In order to successfully build
Original mutation library, for genome rearrangement afterwards, we are finally determined to be used as from diploid Wine brewing yeast strain and set out
Bacterial strain.
Because compared with preserving strains A Y12 and AY15 with laboratory, the bacterial strain separated from industrial production strain
AY12-G has good fermenting property and high temperature resistance, therefore selects strains A Y12-G as the mutagenesis of ARTP ions, gene
Group resets the starting strain being combined with high temperature acclimation in breeding experiment.
ARTP has that easy to operate, cost is low, it is excellent to participate in mutagenic processes etc. without poisonous and harmful substances compared with molecule manipulation
Point, its active-energy particle being rich in can cause to damage to the inhereditary material of bacterial strain, and induce biological cell and start SOS reparations
Mechanism.SOS repair processes are a kind of high serious forgiveness reparation, therefore the abundant mismatch site of species can be produced in repair process, and
Final stable heredity and then formation mutant strain.Because SOS Repair strengths and the DNA degree being damaged have a very big association, and by
Umu-test methods understand that ARTP enriches to biological damage of genetic materials positive effect, damage mechanisms, especially for dye
The Eukaryotic inhereditary material such as colour solid has very strong damage effect, therefore, compared with traditional method of mutagenesis, using ARTP
The multifarious damages of DNA can be effectively caused, mutation rate is high, and easily obtains the good mutant strain of genetic stability.It follows that
ARTP ion mutagenesis is a kind of mutation, rather than the change of the same race for some ad-hoc locations of Yeast genome.It is this
The effective effect eliminated caused by several method of mutagenesis to same position of mutation, ensure that the perfect of mutated library.Therefore
The requirement of genome rearrangement after ARTP ion mutagenesis meets, it can be used for building original mutation library.
Beneficial effect：
The present invention provides a kind of resistant to elevated temperatures Wine brewing yeast strain and its construction method, overcomes common saccharomyces cerevisiae and is coercing
The problem of bad is grown under the conditions of compeling while is of great importance in industrial production to improving fermentation production efficiency.
The present invention carries out ARTP ion mutagenesis, genome rearrangement on the premise of the excellent fermenting property of saccharomyces cerevisiae is ensured
And high temperature acclimation, having obtained one plant of stable thermotolerant ethanol fermentation yeast, cell survival rate and ethanol production increase simultaneously
The present invention use ARTP ion induced-mutation techniques, ARTP compared with molecule manipulation, with it is easy to operate, cost is low, nothing
Poisonous and harmful substances participate in the advantages that mutagenic processes, and its active-energy particle being rich in can cause to damage to the inhereditary material of bacterial strain
Wound, and induce biological cell and start SOS repair mechanisms.SOS repair processes are a kind of high serious forgiveness reparation, therefore in repair process
The abundant mismatch site of species can be produced, and finally stablizes heredity and then forms mutant strain.
Brief description of the drawings：
Fig. 1 is AY12-G growth curve
Fig. 2 is AY12-G fatal rate curve
Fig. 3 attaches most importance to discharge of bacteria strain corn hydrolyzate fermenting experiment
Fig. 4 attach most importance to discharge of bacteria strain simultaneous saccharification and fermentation experiment
Fig. 5 is the growth curve of bacterial strain after domestication
Fig. 6 is that the tolerance of bacterial strain contrasts
Fig. 7 is genome rearrangement operational flowchart
Embodiment：
The present invention is described below by specific embodiment.Unless stated otherwise, technological means used in the present invention
It is method known in those skilled in the art.In addition, embodiment is interpreted as illustrative, it is not intended to limit the present invention
Scope, the spirit and scope of the invention are limited only by the claims that follow.To those skilled in the art, without departing substantially from this
The various changes carried out on the premise of invention spirit and scope to the material component in these embodiments and dosage or change
Belong to protection scope of the present invention.
Embodiment 1：Simultaneous saccharification and fermentation determines parent strain
By Angel high temperature resistant active dry yeast with after the activation of 20g/L syrup, in the areas of YEPD solid plates Shang Hua tri- to obtain
Saccharomyces cerevisiae single bacterium colony is obtained, drawing inclined-plane with the preferable single bacterium colony of transfer needle picking growing way preserves, and is named as AY12-G.Will
AY12-G preserves strains A Y12, AY15 with this laboratory and together carries out maize raw material simultaneous saccharification and fermentation, to select performance most
Parent strain of the excellent bacterial strain as subsequent experimental.After saccharomyces cerevisiae accesses fermentation medium and cultivates 12h under the conditions of 30 DEG C, turn
Enter 35 DEG C, fermented under the conditions of 160r/min.Producing and ethanol ability of each bacterial strain under the conditions of 33 DEG C, 35 DEG C is tested respectively and is gone back
Raw sugar Utilization ability.Under being main fermentation temperature at 33 DEG C, strains A Y12-G producing and ethanol ability be slightly higher than AY12 and
AY15；35 DEG C are that strains A Y12-G producing and ethanol ability is slightly higher than AY15, hence it is evident that higher than AY12 under main fermentation temperature.Two
Under kind main fermentation temperature, the Utilization ability of strains A Y12-G reduced sugar is better than strains A Y12 and AY15, especially 35
Under the conditions of DEG C.In order to filter out the bacterial strain for function admirable of being fermented under higher temperature, selected 35 DEG C of this experiment is used as main fermentation
Temperature.Because AY12 producing and ethanol ability and reduced sugar Utilization ability under the conditions of 35 DEG C is weaker, thus will to AY12-G and
AY15 continues to test, and parent strain is filtered out from two plants of bacterium.Measure AY12-G and AY15 fermented in 33 DEG C of fermentations and 35 DEG C
48h cell survival rates in journey, therefrom to select the more excellent bacterial strain of growth performance.When fermentation carries out 48h, i.e. main fermentation is completed
Afterwards, the zymotic fluid in fermentation flask is shaken up into sampling, after the maize pulp in fermentation sample liquid is removed with three layers of filtered through gauze, sterile washing
The thalline in fermentation sample liquid is washed, after being diluted to suitable multiple, thalline is dyed with methylene blue development process, uses blood count
Plate carries out thalline counting, calculates the cell survival rate of each bacterial strain after the completion of 48h main fermentations either in 33 DEG C or 35 DEG C, 48h
After the completion of main fermentation, strains A Y12-G cell survival rate is above AY15.Producing and ethanol ability and reduced sugar utilize with reference to before
The experimental result of ability, therefore selected parent strains of the strains A Y12-G as subsequent experimental.
The contrast of each bacterial strain producing and ethanol performance and reduced sugar Utilization ability under the different temperatures of table 1
The cell survival rate of each bacterial strain after the completion of the main fermentation of table 2
Technology of alcohol
(1) preparation of corn hydrolyzate
1500g corn flour is weighed, adds 65 DEG C~70 DEG C of 4500mL running water, stands and places 20min, make corn
The abundant water swelling of particle.α-amylase (alpha-amylase) 0.9ml is added, liquefy 1.5h at 85 DEG C~90 DEG C.After the completion of liquefaction
Turn in 60 DEG C of water-baths, and add 3mL carbohydrase, be saccharified 20h at 55 DEG C~60 DEG C.After the completion of saccharification, saccharified liquid is filtered
Cloth filters, and obtains clear filtrate, as corn hydrolyzate, pH 10min naturally, boiling water sterilizes.
(2) corn hydrolyzate hot fermentation
Seed culture medium：8 ° of Brix corn hydrolyzates, 0.5% yeast extract is added, 5mL is in test tube for packing, boils
10min is to sterilize.
Corn hydrolyzate hot fermentation：Preparing 20 ° of Brix corn hydrolyzates, 45mL is in 150mL triangular flasks for packing, and 105
DEG C sterilizing 15min, adds 333 μ L nutritive salt, above-mentioned seed liquor is all accessed, is placed in 35 DEG C, 160r/ after drying in the air to room temperature
Hot fermentation is carried out in min shaking tables.
(3) seed culture medium
Primary-seed medium：YEPD fluid nutrient mediums.
Secondary seed medium：12 ° of Brix of pol molasses culture medium, add 5g/L yeast extracts, 0.5g/L ammonium sulfate.
(4) simultaneous saccharification and fermentation technique
100g corn flour is weighed in the 500mL triangular flasks cleaned, dry and weighed in advance, adds 200g 60 DEG C~70
DEG C running water and 30U/g raw materials amylase, adjust pH to 5.5-5.8, stir, boiling water bath liquefaction, feed temperature reaches
Start timing liquefaction 90min to 90 DEG C, be stirred continuously in liquefaction process, liquefaction result tried using iodine without blueness and in rufous as
It is accurate.It is cooled to 38 DEG C or so after the completion of liquefaction, adjusts pH to 4.2-4.4, adds the carbohydrase of 180U/g raw materials, 0.36g urea,
0.8g yeast cake mud, is stirred, and the water lost in liquefaction process is supplied with 35 DEG C or so of running water, by fermentation flask with general
Logical sealed membrane is sealed, then adds one layer of PE gloves, and rubber band tightly wrapped, and 30 DEG C, 12h is cultivated under the conditions of 160r/min, after be transferred to 35
DEG C, shaker fermentation is carried out under the conditions of 160r/min.
Embodiment 2：ARTP plasma mutagenesis
The ring bacterium mud of picking one is accessed in 5mLYEPD fluid nutrient mediums, 30 DEG C, after 180r/min shaking table cultures 12h, is connect above-mentioned
The μ L of bacterium solution 500 are in the fresh YEPD fluid nutrient mediums of 5mL, 30 DEG C, 180r/min shaking table cultures 4-6h.Appropriate above-mentioned bacterium solution is taken to use
1000 times of normal saline dilution, take the bacterium solution after 10 μ L dilutions to select in carrying out plasma mutagenic treatment on ARTP mutagenesis slide glasses, locate
It is respectively 20s, 30s, 40s, 50s, 60s, 70s to manage the time.After the completion of mutagenic treatment, the slide glass after processing is placed in added with 1mL
In the 2mL centrifuge tubes of sterile saline, whirlpool concussion instrument acutely shakes 3min, takes the bacterium solution after concussion to be coated on YEPD and puts down
Plate, it is placed in 30 DEG C of constant incubators and is cultivated.Draw first round destruction curve, take the death rate >=60% to 100% when
Between section, carry out the measure of next round destruction curve, circulation for several times, until select suitable mutation time.
Suitable mutation time is chosen according to the destruction curve drawn out, mutagenic treatment is carried out to AY12-G.Due to warp
Cross after mutagenic treatment, mutant cell quantity is very little and back mutation easily occurs, therefore the bacterium solution after mutagenesis is accessed new
In fresh YEPD fluid nutrient mediums, the stable culture of a period of time is carried out in 30 DEG C, under conditions of 180r/min, it is prominent to expand
Become the quantity of cell and the stable heredity being mutated.Bacterium solution after culture is diluted to suitable multiple, is applied to YEPD flat boards
On, culture 24h in 37 DEG C of constant incubators is placed in, larger bacterium colony is chosen from flat board and is preserved, filters out mutant bacteria altogether
150 plants of strain.
Embodiment 3：Genome rearrangement
Genome rearrangement is carried out to the 150 plants of bacterium screened after ARTP ion mutagenesis, further to improve the resistance to of bacterial strain
High-temperature behavior.Obtain 150 plants of direct mutation cell colonys are all inoculated into fresh YEPD fluid nutrient mediums, at 30 DEG C,
Cell is collected by centrifugation to logarithmic phase in culture under the conditions of 180 r/min.The thalline being collected into is cleaned with sterilized water, will then be washed
In the net bacterium mud access pre- product spore culture mediums of YPK, at 28 DEG C, 12h is cultivated under the conditions of 180r/min, thalline is collected by centrifugation.With nothing
After bacterium water washs the thalline being collected into three times, in access efficient liquid life spore culture medium, in 28 DEG C, under conditions of 180r/min
Cultivate 5d.Thalline is collected by centrifugation, carries out spore purification afterwards.
Obtained mutagenized populations are first given birth to the thalline after spore to be resuspended in softening buffer solution, incubated under the conditions of 30 DEG C
After 10min, yeast cells and spore is collected by centrifugation, and is resuspended in protoplast buffer, adds 3% glusulase,
And 30min is reacted under the conditions of being placed on 30 DEG C, thalline is collected by centrifugation, thalline is resuspended with 0.5% TritonX-100, afterwards
Ultrasonication is carried out, the continuous scattered situation of observation clasmatosis and spore in processing procedure, to without obvious diploid cell
And spore is collected by centrifugation when being uniformly dispersed in spore.With sterile water washing three times, obtained spore is resuspended in 0.5%
In TritonX-100 solution.
Obtained spore is transferred in YEPD fluid nutrient mediums, at 30 DEG C, 24h is cultivated under conditions of 100r/min, is made
Spore is able to that fully random fusion occurs.Cell is collected, and is coated on YEPD flat boards, is placed in 37 DEG C of incubators and trains
24h is supported, larger bacterium colony is chosen from flat board and preserves and carries out secondary screening, sieves to obtain 137 plant mutant strains altogether.
Fermented in this research using 35 DEG C of corn hydrolyzates, ethanol contains in high performance liquid chromatography (HPLC) measure zymotic fluid
Measure, the method for residual sugar content carries out secondary secondary screening to 137 plant mutant strains in titration determination zymotic fluid.Carrying out corn hydrolyzate hair
In 137 plants of bacterium of ferment, sharing the ethanol production of 14 plants of bacterium has 10%-14% lifting compared with starting strain, and residual sugar content is relatively set out
Bacterial strain has decline by a small margin, i.e., comprehensive fermenting property has been lifted compared with starting strain.By this 14 plants of bacterium be named as L-X and according to
Simultaneous saccharification and fermentation technique, main fermentation temperature is 35 DEG C of progress fermenting experiments, and ethanol production and residual sugar of the measure per bacterial strain contain
Amount.The lifting of comprehensive ethanol production and the reduction of residual sugar content, the relatively good bacterial strain of 7 plants of fermenting properties is selected from 14 plants of bacterium and is made
For the starting strain of subsequent high temperature domestication experiment.This 7 plants of bacterium are respectively L-19, L-25, L-26, L-38, L-90, L-122 and L-
130, the wherein best bacterial strain of fermenting property is L-38, after the completion of its simultaneous saccharification and fermentation, ethanol production 16.2, relatively goes out bacterium germination
Strain AY12-G ethanol production improves 8%, and residual sugar content is 35.5g/L, and the residual sugar content compared with AY12-G reduces
32.38%.
Embodiment 4：High temperature acclimation
The selection of acclimation temperature
To ensure being smoothed out for high temperature acclimation experiment, we have carried out the culture experiment of thermograde to AY12-G, with
Search out suitable acclimation temperature.A ring starting strain AY12-G is met in fresh YEPD culture mediums, in 30 DEG C, 180r/min
Under the conditions of cultivate 12h, obtain primary seed solution, primary seed solution accessed in several bottles of fresh YEPD culture mediums, controls its initial
OD600For 2.00, shaking flask is respectively placed in 38 DEG C, 40 DEG C, 42 DEG C, 44 DEG C, cultivated in 180r/min condition, observation is each
Bacterial concentration reaches OD under individual growth temperature600The time consumed during by 10.00.As a result it is as shown in table 3.
The growing states of the AY12-G of table 3 at various temperatures
It can be seen from table, for starting strain AY12-G, high temperature has certain inhibitory action to its growing state, with
The rise of growth temperature, growth the consumed time is also increasingly longer, because the Wine brewing yeast strain after breeding is final
Industrial production need to be applied to, and be 33 DEG C according to the conventional fermentation temperature recognized from industrial production, conducted in this research
First run acclimation temperature elect 41 DEG C as, two wheel acclimation temperatures are 42 DEG C.
Reset the high temperature acclimation of bacterial strain
By select bacterial strain L-19, L-25, L-26, L-38, L-90, L-122 and L-130 after genome rearrangement points
In YEPD fluid nutrient mediums that Jie Ru be not fresh 5mL, in 30 DEG C, 12h is cultivated under the conditions of 180r/min, obtains the one of each bacterial strain
Level seed liquor.By in shaking flasks of the primary seed solution access 150mL equipped with the fresh YEPD fluid nutrient mediums of 50mL of each bacterial strain, adjust
Save the initial OD of each shaking flask bacterium solution600For 2.0, shaking flask is placed in 41 DEG C, is cultivated, is treated in shaking flask in 160r/min shaking tables
Bacterial concentration reach OD600For 10.0 when, the bacterium solution in shaking flask is accessed to fresh YEPD fluid nutrient mediums again, put again
Enter 41 DEG C, the culture domestication of a new round is carried out in 160r/min shaking tables.
When the consumed time of growth under growth temperature of each bacterial strain at 41 DEG C foreshortening to 1 day, by the bacterium in shaking flask
Liquid accesses fresh YEPD fluid nutrient mediums again, and acclimation temperature is adjusted into 42 DEG C, carries out the domestication of the second wheel.
After the completion of the domestication for treating the second wheel, the bacterium solution after each bacterial strain is tamed is diluted to suitable multiple, is coated on YEPD
Flat board, flat board is placed in 37 DEG C of constant incubators and cultivated, when single bacterium colony is grown on flat board, therefrom chosen bigger
Single bacterium colony is preserved.It is X-19, X-25, X-26, X-38, X-90, X-122 and X-130 by the Strain Designation after domestication.
Embodiment 5：The measure of growth curve
This experiment is using full-automatic growth curve analyzer measure OD600Light absorption value, operating procedure are as follows：
(1) ring of picking slant strains 1 is connected in 5mLYEPD fluid nutrient mediums, 30 DEG C, 180r/min cultures 12h.
(2) hole of above-mentioned cultured 100 orifice plates of the μ L of bacterium solution 40 accesses equipped with 360 μ L liquid YEPD culture mediums is drawn
In, cultivate at a temperature of 100 orifice plates are placed in into setting, using YEPD fluid nutrient mediums as blank control, determined every 0.5h
Light absorption value at 600nm, using the time as abscissa, OD600It is worth for ordinate, measure high temperature acclimation strain X -19, X-25, X-
26th, the growth curve of X-38, X-90, X-122, X-130 and starting strain AY12-G under the conditions of 36 DEG C, to compare naturalized strain
With the growth performance of starting strain.When tame complete after, each bacterial strain is placed under the conditions of 36 DEG C when being grown, with setting out
Strains A Y12-G is compared, and the growth performance of each bacterial strain has certain difference.Wherein, X-19 and X-38 is under the conditions of 36 DEG C
Growth performance is similar to AY12-G, into stationary phase time slightly have after prolong, but final bacterium is dense but more slightly higher than starting strain；Bacterium
Strain X-25, X-26 is similar with X-130 growth performance, but compared with AY12-G, the time into stationary phase is slower, with X-19
Compared with X-38, the time into stationary phase slightly shifts to an earlier date, and final cell concentration is similar to starting strain；Strain X -90 and X-
120 growth performance is similar, and compared with other bacterial strains, difference is more obvious, and the time consumed into stationary phase is longer, most
Whole cell concentration has also declined.All things considered, it have passed through the bacterium after ARTP mutagenesis, genome rearrangement and high temperature acclimation
Strain, growth performance have certain change or increase or reduce but do not influence overall fermentation process.
Embodiment 6：The measure of saccharomyces cerevisiae corn hydrolyzate fermentation parameter
The content of the second alcohol and glucose in zymotic fluid is determined using high performance liquid chromatography (HPLC).HPLC detector bar
Part：Bio-Rad HPX-87H chromatographic columns, mobile phase are 5mmol/L sulfuric acid, flow velocity 0.6mL/min, 65 DEG C of column temperature, are detected
Device temperature is 45 DEG C, is detected using differential refraction detector.Sample is diluted to the membrane filtration with 0.22 μm after certain multiple,
Sample size is 20 μ L.External standard method is used to be quantified with the peak height of chromatogram.All standard curve R2Value reaches 0.999 with top
It can be used.
The fermenting property and survival rate of the naturalized strain of table 4 and active dry yeast
As can be seen from Table 3 compared with Angel high temperature resistant active dry yeast, the bacterial strain after domestication omits on ethanol production
There is lifting, residual sugar content has also declined accordingly, CO2Total weight loss equally increased accordingly, but fermentation time has extension,
Main fermentation hair is into rear, i.e., cell is substantially higher in 48h survival rate than Angel high temperature resistant active dry yeast, naturalized strain X-19,
X-25, X-26, X-122 and X-130 ratio respectively improve 2.12 times, 2.00 times, 1.95 times, 2.22 times and 2.00 times, explanation
Bacterial strain 48h cell survival rates after domestication are higher than Angel high temperature resistant active dry yeast.
Embodiment 7：Brewing yeast cell tolerance qualitative determination
(1) from the ring yeast bacterium mud of inclined-plane picking one, access in 5mL YEPD fluid nutrient mediums, 30 DEG C, 180r/min is overnight
Culture.Survey OD600Value, take appropriate bacterium solution to transfer in the fresh YEPD fluid nutrient mediums of 5mL, make its initial OD600It is worth for 0.15,
Cultivate certain time.
(2) OD is measured600Value, cell OD is adjusted with YEPD fluid nutrient mediums600=1.
(3) 100 μ L bacterium solution is pipetted respectively with liquid-transfering gun in 1.5mL centrifuge tubes, and experimental group does the processing of water-bath thermal shock, right
It is without any processing according to organizing.
(4) experimental group and control group are diluted into identical concentration gradient with sterilized water respectively, according to the order of descending concentrations,
Each dilution factor takes the neat drop of 2 μ L bacterium solution, and on YEPD solid medium flat boards, after super-clean bench dries, sealed membrane is sealed down
It is placed in 30 DEG C of constant incubators and cultivates 1-3d, observe the growth of thalline, compares the heat-resisting situation of different strains.
(5) the neat drop of bacterium solution of 2 μ L control groups is pipetted on the YEPD culture medium flat plates of the NaCl containing 80g/L, is surpassed
After net platform dries, sealed membrane, which is sealed to be inverted in 30 DEG C of constant incubators, cultivates 1-3d, observes thalli growth, compares different strains
Tolerated.
On the YEPD flat boards of no growth pressure, starting strain is substantially consistent with the growing state of naturalized strain
, it is seen that it is substantially consistent to be used as the starting strain of tolerance test and the initial cell concentration of naturalized strain.56
Under environmental pressure caused by the processing of DEG C (3min) thermal shock, starting strain AY12-G and naturalized strain X-19, X-25, X-26, X-
38th, X-90, X-122 and X-130 growth, which receive, significantly suppresses, in addition to X-122, other naturalized strains
Thermal shock resistance is better than starting strain, and wherein X-25, X-26 and X-130 thermal shock resistance is better than remaining naturalized strain；
Under environmental pressure caused by 80g/L NaCl, starting strain and naturalized strain all receive a certain degree of suppression, tame bacterium
Strain X-19, X-25, X-26, X-38, X-90, X-120 salt tolerance are better than starting strain AY12-G, wherein X-26 salt tolerant
Property is better than the salt tolerance of other naturalized strains, and naturalized strain X-130 salt tolerance is poorer than AY12-G.Summary synchronous fermentation
As a result and tolerance measurement result finally filters out the results strain that one plant of X-130 tests as this.
Claims (6)
1. the thermotolerant ethanol fermentation yeast selected, it is arriving by the mutagenesis of ARTP ions, genome rearrangement and high temperature acclimation.
2. thermotolerant ethanol fermentation yeast bacterial strain as claimed in claim 1, it is characterised in that the starting strain is saccharomyces cerevisiae
(Saccharomyces cerevisiae)CICC32315。
3. thermotolerant ethanol fermentation yeast bacterial strain as claimed in claim 1, it is characterised in that described saccharomyces cerevisiae has normal
Growth and fermenting property, and to high temperature, salt has the yeast strain of certain tolerance.
4. the construction method of thermotolerant ethanol fermentation yeast bacterial strain as claimed in claim 1, it is characterised in that the construction method is
The method being combined by ARTP orientation ion mutagenesis and genome rearrangement, select the stable yeast strain AY12-G of one plant of character
As starting strain by mutagenesis and gene rearrangement obtain one plant of resistant to elevated temperatures yeast strain then to its income high temperature acclimation most
Afterwards to results strain cell survival rate and fermenting property be better than starting strain.
5. the construction method of high tolerance Wine brewing yeast strain as claimed in claim 4, it is characterised in that specific steps include：
By 35 DEG C of simultaneous saccharification and fermentations, the Wine brewing yeast strain conduct obtained by separation screening in industrial strain finally have selected
The parent strain of molecular modification, it is named as AY12-G strains；
It is handled successively using the mutagenesis of traditional breeding method means ARTP ions, genome rearrangement and high temperature acclimation；
After ion mutagenesis, genome rearrangement, finally select the excellent bacterial strain of 7 plants of fermenting properties and carry out high temperature acclimation；
After domestication terminates, by growth performance, 35 DEG C of simultaneous saccharification and fermentation results and the 48h under the conditions of 36 DEG C of naturalized strain
The measure of cell survival rate understands that the 48h cell survival rates of bacterial strain have been lifted, but fermenting property does not change significantly,
On the contrary, fermentation period has extended.After acidic protein ferment treatment is with the addition of to fermentation medium, the fermentation period of naturalized strain
Shorten.Comprehensive Experiment result, it is final to obtain that fermenting property is more excellent, and the higher strain X of 48h cell survival rates-
130。
6. application of the thermotolerant ethanol fermentation yeast bacterial strain as claimed in claim 2 in alcohol production.
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| CN108841736A (en) * | 2018-04-19 | 2018-11-20 | 江南大学 | It is a kind of with the ethyl alcohol thick mash fermentation superior strain of multiple tolerance and its application |
| CN111334442A (en) * | 2018-12-19 | 2020-06-26 | 吉林中粮生化有限公司 | High-temperature-resistant saccharomyces cerevisiae strain and application thereof |
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| CN103232948A (en) * | 2013-05-10 | 2013-08-07 | 天津科技大学 | High-temperature resistant saccharomyces cerevisiae strain and breeding method thereof |
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| CN103232948A (en) * | 2013-05-10 | 2013-08-07 | 天津科技大学 | High-temperature resistant saccharomyces cerevisiae strain and breeding method thereof |
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| CN108841736A (en) * | 2018-04-19 | 2018-11-20 | 江南大学 | It is a kind of with the ethyl alcohol thick mash fermentation superior strain of multiple tolerance and its application |
| CN108841736B (en) * | 2018-04-19 | 2020-09-04 | 江南大学 | High-yield strain with multiple tolerance for ethanol thick mash fermentation and application thereof |
| CN111334442A (en) * | 2018-12-19 | 2020-06-26 | 吉林中粮生化有限公司 | High-temperature-resistant saccharomyces cerevisiae strain and application thereof |
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