CN105368729A - Acid-tolerant flocculation industrial Saccharomyces cerevisiae strain and construction method - Google Patents
Acid-tolerant flocculation industrial Saccharomyces cerevisiae strain and construction method Download PDFInfo
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Abstract
The invention discloses an acid-tolerant flocculation industrial Saccharomyces cerevisiae strain and a construction method and solves the problem that most strains fused by existing fusion techniques only effectively have inheritable characters of both parents and cannot effectively improve the inheritable character of fused strains. The invention includes a Saccharomyces cerevisiae strain SEB4, collected in China General Microbiological Culture Collection Center under CGMCC11324. The invention also provides a construction method of the Saccharomyces cerevisiae strain SEB4. The Saccharomyces cerevisiae strain SEB4 fused has inheritable characters of both parents, has good flocculability and fermenting capacity as in a parent fusant 1 and good acid tolerance as in a parent SEB2, and is superior to a parent strain both in ethanol yield and glucose consumption rate.
Description
Technical field
The present invention relates to a kind of yeast strain, what be specifically related to is the construction process of a strain acidproof flocculence an industrial strain of S.cerevisiae strain and this bacterial strain.
Background technology
Biofuel ethanol is considered to the important energy source of alternative conventional fuel oil.Yeast saccharomyces cerevisiae is the main microorganism used in bio-ethanol industrial production, and the optimum pH of general bacterial strain is 4-5.When pH is higher or lower than this scope, the growth of bacterial strain and fermentation capacity decline.And large-scale industrial production needs well to control living contaminants, the pH value as far as possible reducing fermentation system compares effective measures.In addition, lignocellulose-like biomass is important fuel ethanol production raw material, and diluted acid need be adopted to carry out pre-treatment to the first step that it utilizes, consequent hydrolyzed solution pH value is lower, could be used for the ethanol fermentation of yeast after needing to be adjusted to suitable pH value.Therefore, the quality yeast bacterial strain obtaining tolerance low ph value contributes to the stability improving alcohol production, reduces production cost.
Protoplast fusion is a kind of conventional Microbial Breeding technology, refers to by artificial method, and the protoplastis of two cells making inherited character different merges, so as to obtaining the process having the stable recon of amphilepsis proterties concurrently.Compared with other breeding techniques, Protoplast Fusion Technique have recombination frequency higher, to educate the advantages such as the less and genetic material of type restriction is more complete by mating type or cause, thus by domestic and international Microbial Breeding scholar widespread use.
Summary of the invention
The object of the invention is to solve existing integration technology merge the bacterial strain and mostly can only effectively have amphilepsis proterties concurrently, and can not effectively improve merge after the problem of bacterial strain inherited character performance; There is provided a strain effectively can have amphilepsis proterties concurrently, on ethanol production, glucose utilization and wear rate, all can also be better than the acidproof flocculence an industrial strain of S.cerevisiae strain of parent strain simultaneously, and provide the construction process of this bacterial strain.
For achieving the above object, concrete technical scheme of the present invention is as follows:
The strain of one strain acidproof flocculence an industrial strain of S.cerevisiae, described yeast saccharomyces cerevisiae (
saccharomycescerevisiaesEB4) bacterial strain is preserved in China General Microbiological culture presevation administrative center, and preserving number is CGMCC11324, and the time is on September 6th, 2105.
Saccharomyces Cerevisiae in S EB4 bacterial strain after merging in the present invention, not only has amphilepsis proterties concurrently, namely has the good flocculence of the sub-RHZ-1 of parent fusion and fermentation capacity, and the acid-fast ability that parent SEB2 is good; And it is all better than parent strain on ethanol production, glucose utilization and wear rate.
Prove through experiment, at 30 ° of C, pH2.2, under to enter sugared concentration be 150g/l condition:
During 24h, Saccharomyces Cerevisiae in S EB4 bacterial strain sugar consumption amount is 78.7%, sugar consumption speed is 4.9g/l/h, ethanol production is 47.87g/l, parent fusion RHZ-1 sugar consumption amount is 64.9%, sugar consumption speed is 4.1g/l/h, ethanol production is 35.93g/l, and parent SEB2 sugar consumption amount is 71.6%, sugar consumption speed is 4.5g/l/h, ethanol production is 32.69g/l;
During 48h, glucose all can all consume by all bacterial strains, and Saccharomyces Cerevisiae in S EB4 bacterial strain ethanol production is 57.76g/l, and the sub-RHZ-1 ethanol production of parent fusion is 51.93g/l, and parent SEB2 ethanol production is 54.42g/l.
The construction process of Wine brewing yeast strain as claimed in claim 1, comprises the following steps:
1) structure of auxotrophic mutation bacterium
1.1) with yeast saccharomyces cerevisiae
saccharomycescerevisiaeiR-2 and yeast saccharomyces cerevisiae
saccharomycescerevisiaesEB1 is parent, obtains fusant RHZ-1 by cytogamy mode;
Wherein, SEB1 bacterial strain, has ethanol production, is preserved in China General Microbiological culture presevation administrative center, and deposit number is CGMCC11321.IR-2 bacterial strain: be separated in Indonesia's leavened food, there is ethanol production, there is flocculence, source is documented in following document: HiroshiK, YoshioS, ToshioM, HarumiK, YorikazuS.1985.Continuousethanolfermentationwithcellrecy clingusingflocculatingyeast.J.Ferment.Technol.63:159-165; Name after document translation is called: use flocculence yeast to carry out being with the continuous ethanol fermentation of cell cycle; Above-mentioned IR-2 biomaterial obtains from Japanese industries Technical Integration Studies institute (NationalInstituteofAdvancedIndustrialScienceandTechnolog y, AIST).The fusion method of above-mentioned IR-2 bacterial strain and SEB1 bacterial strain is as follows:
SEB1 and IR-2 is at product Spore cultivation base (0.5% Lithium Acetate, 2% agar) go up growth 3 days, the ascus of gained obtains spore through the process of yeast lyase, 2 hours are processed through ethylmethane sulfonate (EMS), YPD slat chain conveyor is coated onto after ultrasonic disperse, then photocopy was to minimum nutrition grow on plates 4 days, checked the auxotroph of the bacterium colony do not grown on minimum nutrition flat board.Obtain the bacterial strain SIV-2 of Isoleucine and α-amino-isovaleric acid defective type from SEB1, obtain the bacterial strain IL-1 of lysine deficient from IR-2.
The formation of above-mentioned YPD flat board is: 2% glucose, 1% yeast powder, 2% polyprotein peptone, 2% agar, and all the other are distilled water; The formation of above-mentioned minimum nutrition flat board is: 2% glucose, and 0.67% without amino acid yeast nitrogen, 2% agar, and all the other are distilled water.
SIV-2 and IL-1 is through the process of yeast lyase after 2 hours, and the protoplastis obtained mixes process 15 minutes in the PEG6000 of 30%, is coated onto regenerated plate and selects on flat board.Selecting the bacterium colony of grow on plates to be considered as fusant, fusant quantity is fusion efficiencies with the plasmic quantity ratio of regeneration.Obtaining 8 strain fusant RHZ-1(fusion efficiencies is altogether 1.1 × 10
-5), screening acquisition utilizes the fusant RHZ-1 of 25% molasses fermented producing and ethanol the fastest (1.3g/l/h) and alcohol concn the highest (65g/l).
The formation of above-mentioned regenerated plate is: 2% glucose, 0.5% yeast powder, 1% polyprotein peptone, 4.5% Repone K, 2% agar, and all the other are distilled water; The formation of above-mentioned selection flat board is: 2% glucose, and 0.67% without amino acid yeast nitrogen, 2% agar, and all the other are distilled water.
1.2) fusant RHZ-1 and SEB2 is carried out ultraviolet mutagenesis, filter out the single auxotrophic mutation bacterium of fusant RHZ-1 and SEB2 respectively; SEB2 is deposited in China General Microbiological culture presevation administrative center, and deposit number is CGMCC11322.
1.3) from fusant RHZ-1 single auxotrophic mutation bacterium, the good bacterial strain of flocculence is filtered out, and energy for growth and leavening property evaluation experimental are carried out to this bacterial strain, pick out the auxotrophic mutation bacterium KFU of bacterial strain as subsequent native plast fusion of leavening property optimum; Energy for growth and leavening property evaluation experimental are carried out to SEB2 single auxotrophic mutation bacterium, filters out the auxotrophic mutation bacterium KAGOU of bacterial strain as subsequent native plast fusion of best performance;
2) structure of Protoplast fusant
2.1) using auxotrophic mutation bacterium KFU and auxotrophic mutation bacterium KAGOU as parent, carry out the fusion of protoplasm somatocyte, obtain fusant RHZ-2;
2.2) by the suspension after merging on flat board, screen the fusant RHZ-2 grown, filter out and have flocculence and acid-fast ability and the bacterial strain of leavening property the best simultaneously, this bacterial strain is Saccharomyces Cerevisiae in S EB4.
The present invention is with 2 industrial saccharomyces cerevisiaes for parent strain, and in conjunction with ultraviolet mutagenesis and Protoplast Fusion Technique, obtain the good acidproof flocculence yeast strain of 1 strain leavening property, the production that can be alcohol fuel provides excellent Microbial resources.The bacterial strain that method of the present invention constructs, not only has amphilepsis proterties concurrently, namely has the good flocculence of the sub-RHZ-1 of parent fusion and fermentation capacity, and the acid-fast ability that parent SEB2 is good; And effectively can also improve the rear bacterial strain inherited character performance of fusion, the fusant bacterial strain that namely method of the present invention builds all is better than parent strain on ethanol production, glucose utilization and glucose consumption rate.
Further, the specific operation process of described ultraviolet mutagenesis is as follows:
Proceeded to respectively by fusant RHZ-1 and SEB2 in 2%YPD substratum, 30 ° of C activate 16h; Bacterium liquid is applied on YPD flat board after sterilized water dilution, after flat board irradiates 50s under ultraviolet lamp, in 30 ° of C quiescent culture.
Further, the screening process of described single auxotrophic mutation bacterium is as follows:
The bacterial strain of the YPD slat chain conveyor through ultraviolet lamp process is fallen photocopy on MM flat board, and in 30 ° of C incubated overnight, the bacterium colony that contrast YPD and MM flat board grows, picking out from YPD flat board cannot at the bacterium colony of MM plated growth, i.e. auxotrophic mutation bacterium;
Add different nutritive substances to MM flat board, each dull and stereotyped a kind of nutrition, cultivates auxotrophic mutation bacterium Simultaneous vaccination, identifies its auxotrophy type, and filter out single auxotrophic mutation bacterium to YPD and MM flat board.
Wherein, 2%YPD substratum comprises: 20g/l glucose, 10g/l yeast extract powder, 20g/l peptone, and all the other are distilled water; MM substratum comprises: 1.7g/l is without amino yeast nitrogen, 10g/l yeast powder, 20g/l glucose, and all the other are distilled water.The dull and stereotyped agar that with the addition of 20g/l on the basis of substratum.
The present invention compared with prior art, has the following advantages and beneficial effect:
Saccharomyces Cerevisiae in S EB4 bacterial strain after merging in the present invention, not only has amphilepsis proterties concurrently, namely has the good flocculence of the sub-RHZ-1 of parent fusion and fermentation capacity, and the acid-fast ability that parent SEB2 is good; And it is all better than parent strain on ethanol production, glucose utilization and glucose consumption rate.
The preservation information relating to the microorganism of preservation in the present invention is as follows:
Depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center; Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica; Preservation date: on September 6th, 2015; Deposit number: CGMCC11321, CGMCC11322, CGMCC11324; Classification And Nomenclature: Saccharomycescerevisiae.
Accompanying drawing explanation
Fig. 1 is the growth result schematic diagram of auxotrophic mutation bacterium when adding different nutritive substances.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.
Embodiment 1
The strain of one strain acidproof flocculence an industrial strain of S.cerevisiae, described yeast saccharomyces cerevisiae (
saccharomycescerevisiaesEB4) bacterial strain is preserved in China General Microbiological culture presevation administrative center, and preserving number is CGMCC11324.The concrete construction process of this Wine brewing yeast strain SEB4 is as follows:
1) structure of auxotrophic mutation bacterium
1.1) with yeast saccharomyces cerevisiae
saccharomycescerevisiaeiR-2 and yeast saccharomyces cerevisiae
saccharomycescerevisiaesEB1 is parent, obtains fusant RHZ-1 by cytogamy mode.
In the present embodiment, this SEB1 bacterial strain, has ethanol production, is preserved in China General Microbiological culture presevation administrative center, and deposit number is CGMCC11321.IR-2 bacterial strain: be separated in an Indonesian leavened food, there is ethanol production, there is flocculence, source is documented in following document: HiroshiK, YoshioS, ToshioM, HarumiK, YorikazuS.1985.Continuousethanolfermentationwithcellrecy clingusingflocculatingyeast.J.Ferment.Technol.63:159 – 165; Name after document translation is called: use flocculence yeast to carry out being with the continuous ethanol fermentation of cell cycle; Above-mentioned IR-2 biomaterial obtains from Japanese industries Technical Integration Studies institute (NationalInstituteofAdvancedIndustrialScienceandTechnolog y, AIST).The fusion method of above-mentioned IR-2 bacterial strain and SEB1 bacterial strain is as follows:
SEB1 and IR-2 grows 3 days on product Spore cultivation base, the ascus of gained obtains spore through the process of yeast lyase, 2 hours are processed through ethylmethane sulfonate (EMS), YPD slat chain conveyor is coated onto after ultrasonic disperse, then photocopy was to minimum nutrition grow on plates 4 days, checked the auxotroph of the bacterium colony do not grown on minimum nutrition flat board.Obtain the bacterial strain SIV-2 of Isoleucine and α-amino-isovaleric acid defective type from SEB1, obtain the bacterial strain IL-1 of lysine deficient from IR-2.
SIV-2 and IL-1 is through the process of yeast lyase after 2 hours, and the protoplastis obtained mixes process 15 minutes in the PEG6000 of 30%, is coated onto regenerated plate and selects on flat board.Selecting the bacterium colony of grow on plates to be considered as fusant, fusant quantity is fusion efficiencies with the plasmic quantity ratio of regeneration.Obtaining 8 strain fusant RHZ-1(fusion efficiencies is altogether 1.1 × 10
-5), screening acquisition utilizes the fusant RHZ-1 of 25% molasses fermented producing and ethanol the fastest (1.3g/l/h) and alcohol concn the highest (65g/l).
The formation of above-mentioned product Spore cultivation base is: 0.5% Lithium Acetate, 2% agar, and all the other are distilled water; The formation of above-mentioned YPD flat board is: 2% glucose, 1% yeast powder, 2% polyprotein peptone, 2% agar, and all the other are distilled water; The formation of above-mentioned minimum nutrition flat board is: 2% glucose, and 0.67% without amino acid yeast nitrogen, 2% agar, and all the other are distilled water.
The formation of above-mentioned regenerated plate is: 2% glucose, 0.5% yeast powder, 1% polyprotein peptone, 4.5% Repone K, 2% agar, and all the other are distilled water; The formation of above-mentioned selection flat board is: 2% glucose, and 0.67% without amino acid yeast nitrogen, 2% agar, and all the other are distilled water.
1.2) fusant RHZ-1 and SEB2 is carried out ultraviolet mutagenesis, filter out the single auxotrophic mutation bacterium of fusant RHZ-1 and SEB2 respectively; SEB2 is deposited in China General Microbiological culture presevation administrative center, and deposit number is CGMCC11322.Detailed process is as follows:
First, proceeded to respectively by fusant RHZ-1 and SEB2 in 2%YPD substratum, 30 ° of C activate 16h; Bacterium liquid is applied on YPD flat board after sterilized water dilution, after flat board irradiates 50s under ultraviolet lamp, in 30 ° of C quiescent culture.
Secondly, the bacterial colony photographic reprinting grown by flat board after quiescent culture 2d is dull and stereotyped to MM, and in 30 ° of C incubated overnight.The bacterium colony that contrast YPD and MM flat board grows, picking out from YPD flat board cannot at the bacterium colony of MM plated growth, i.e. auxotrophic mutation bacterium.Through 4 repeated screenings checking, the final auxotrophic strain obtaining 3 strain fusant RHZ-1,2 clones are selected in every strain, respectively called after: KFU-A1, KFU-A2; KFU-B1, KFU-B2; KFU-C1, KFU-C2; Obtain the auxotrophic strain of 5 strain SEB2,2 clones are selected in every strain, respectively called after: KAGOU-A1, KAGOU-A2; KAGOU-B1, KAGOU-B2; KAGOU-C1, KAGOU-C2; KAGOU-D1, KAGOU-D2; KAGOU-E1, KAGOU-E2.
Finally, different nutritive substances is added in MM flat board, each dull and stereotyped a kind of nutrition, totally 22 kinds, as shown in table 1, cultivated to YPD and MM flat board by auxotrophic mutation bacterium Simultaneous vaccination, cultivation results as shown in Figure 1, being the auxotrophic strain of fusant RHZ-1 in Fig. 1 (a), is the auxotrophic strain of SEB2 in Fig. 1 (b).
Shown by Fig. 1: KFU-A1, KFU-A2 and KAGOU-D1, KAGOU-D2 is 4 strain uracil-deficient type bacterial strain (Ura
-); KFU-B1, KFU-B2 are 2 strain lysine deficient bacterial strain (Lys
-); KFU-C1, KFU-C2 are 2 strain halfcystine deficient strain (Cys
-); KAGOU-B1, KAGOU-B2 are 2 strain Adenine nucleotides bacterial strain (Ade
-); KAGOU-E1, KAGOU-E2 are 2 strain histidine deficient bacterial strain (His
-); KAGOU-A1, KAGOU-A2, KAGOU-C1, KAGOU-C2 all do not grow on all MM flat boards.
Shown by the above results: this 4 strain bacterium of KAGOU-A1, KAGOU-A2, KAGOU-C1 and KAGOU-C2 is two or more Different Nutrition deficient strain, cannot be used for the screening of Protoplast Fusant, follow-uply no longer it to be investigated.
The single auxotrophic mutation bacterium that the present embodiment filters out is: KFU-A1, KFU-A2, KFU-B1, KFU-B2, KFU-C1 and KFU-C2; And KAGOU-B1, KAGOU-B2, KAGOU-D1, KAGOU-D2, KAGOU-E1 and KAGOU-E2.
Table 1
Wherein, 2%YPD substratum: 20g/l glucose, 10g/l yeast extract powder, 20g/l peptone.MM substratum: 1.7g/l without amino yeast nitrogen, 10g/l yeast powder, 20g/l glucose.Flat board is the agar that with the addition of 20g/l on the basis of substratum.
1.3) parent strain as subsequent native plast fusion is filtered out
1.3.1) bacterial strain that in fusant RHZ-1 single auxotrophic mutation bacterium, flocculence is good is filtered out, and energy for growth and leavening property evaluation experimental are carried out to this bacterial strain, pick out the auxotrophic mutation bacterium KFU of bacterial strain as subsequent native plast fusion of leavening property optimum; Detailed process is as follows:
Fusant RHZ-1 single auxotrophic mutation bacterium is seeded in 2%YPDU substratum and cultivates 16h, get 5ml bacterial culture fluid respectively in test tube, even through vortex oscillation, the flocculence of bacterial strain is observed after leaving standstill different time, find except bacterial strain KFU-A2 through observing, other bacterial strain all remains with the good flocculence of the sub-RHZ-1 of parent fusion.Described 2%YPDU substratum comprises: 2g/l peptone, 1g/l ammonium sulfate, 20g/l glucose, 0.04g/l uridylic, and all the other are distilled water.
Therefore, all bacterial strains energy for growth in acid condition except bacterial strain KFU-A2 is investigated; Energy for growth evaluation procedure is: be seeded in the 2%YPDU substratum of pH3.0 and pH2.7 respectively by fusant RHZ-1 mutant strain, and instrument (ADVANTEC is cultivated in small test tubes under 35 ° of C conditions, Japan) middle cultivation, instrument records the OD that different pH value hypothallus grows 24 hours automatically
660value; Result is as shown in table 2.
Table 2
Known by table 2, bacterial strain KFU-A1, KFU-B1, KFU-B2 under pH3.0 and pH2.7 condition, energy for growth all with parent fusion RHZ-1 bacterial strain close; The growth of bacterial strain KFU-C1 and KFU-C2 has part to suppress, but the growth of KFU-C1 is seriously suppressed under pH2.7 condition.
Therefore, select bacterial strain KFU-A1, KFU-B2 and KFU-C2 carries out leavening property evaluation.
The concrete evaluation procedure that leavening property is evaluated is as follows: be seeded to by the single auxotrophic mutant strain of sub-for parent fusion RHZ-1 in 5%YPDU substratum (pH nature) respectively, and in 30 ° of C, 160rpm (shaking table vibration) preculture 16h.Inoculate in the 15%YPDU substratum of pH2.7, to ferment 48h in 35 ° of C, 400rpm (magnetic agitation), regularly get fermented liquid and detect total cell count, glucose concn and alcohol concn.Following result is obtained: bacterial strain KFU-B2 (Lys after being evaluated by leavening property
-) there is higher leavening property, it is at cell survivaling number, ethanol production, and glucose consumption aspect is better than other bacterial strains.Described 5%YPDU substratum: the glucose concn in 2%YPDU substratum is adjusted to 50g/l, all the other components are constant; Described 15%YPDU substratum: the glucose concn in 2%YPDU substratum is adjusted to 150g/l, all the other components are constant.
To sum up, thus select KFU-B2 as the parent strain of subsequent native plast fusion.
1.3.2) energy for growth and leavening property evaluation experimental are carried out to SEB2 single auxotrophic mutation bacterium, filter out the auxotrophic mutation bacterium KAGOU of bacterial strain as subsequent native plast fusion of best performance; Detailed process is as follows:
With parent strain SEB2 in contrast, adopt the energy for growth evaluation procedure identical with above-mentioned fusant RHZ-1 bacterial strain, compare the growth velocity μ of each mutant strain of SEB2
max(h
-1), OD
660reach 0.5 required time t
oD660=0.5and the maximum OD reached (min)
max, concrete outcome is as shown in table 3.
Table 3
Known by table 3: under pH3.0 and pH2.7 condition, energy for growth and the parent SEB2 of bacterial strain KAGOU-E1, KAGOU-E2 are similar.And the growth of other bacterial strains is subject to certain suppression, when pH is 2.7, suppress situation more obvious.
Meanwhile, select bacterial strain KAGOU-B2, KAGOU-D2 and KAGOU-E2 carries out leavening property evaluation.Namely by the leavening property evaluation procedure identical with above-mentioned fusant RHZ-1 bacterial strain, contrast with parent strain SEB2, fermenting experiment shows: KAGOU-E2 (His
-) at cell survivaling number, ethanol production, glucose consumption aspect, all has good performance than all the other two bacterial strains.
To sum up, therefore select KAGOU-E2 as the parent strain of subsequent native plast fusion.
2) structure of Protoplast fusant
2.1) with auxotrophic mutation bacterium KFU-B2 (Lys
-) and auxotrophic mutation bacterium KAGOU-E2 (His
-) as parent, carry out the fusion of protoplasm somatocyte, obtain fusant RHZ-2;
Protoplastis is prepared in process, and the Protoplast calli that the protoplast formation rate recording two bacterial strains all reaches 100%, KFU-B2 and KAGOU-E2 is respectively 4.0% and 7.5%, shows that breaking yeast cellule membrane is ideal, is conducive to next step and merges.
2.2) by merge after suspension on flat board, the fusant RHZ-2 grown is screened, obtains 16 strain fusant bacterial strains altogether, called after SEB4 ~ 11, F2-1 ~ F2-8.Flocculence is all investigated to above-mentioned 16 strain fusant bacterial strains, has shown that all fusant bacterial strains all have the flocculence of the sub-RHZ-1 equal extent of parent fusion.Meanwhile, under 30 ° of C acidic conditionss, all fusant bacterial strains all can glucose fermentation producing and ethanol, shows that they have been endowed the acid-resistant property of parent SEB2.
Namely the present invention had both had the flocculence of the sub-RHZ-1 equal extent of parent fusion, also had the acid-resistant property of parent SEB2.
The present invention carries out leavening property detection to above-mentioned 16 strain fusant bacterial strains, proves by experiment: the leavening property of bacterial strain SEB4 is best, and it is all better than parent strain on ethanol production, glucose utilization and glucose consumption rate.The concrete leavening property result of bacterial strain SEB4 is as follows:
During 24h, bacterial strain SEB4 consumes maximum glucose, and residual sugar amount is 31.88g/l (sugar consumption amount is 78.7%, and sugar consumption speed is 4.9g/l/h), and ethanol production is 47.87g/l.
And now, the residual sugar amount of the sub-RHZ-1 of parent fusion and parent SEB2 is respectively 52.7g/l, and (sugar consumption amount is 64.9%, sugar consumption speed is 4.1g/l/h) and 42.51g/l (sugar consumption amount is 71.6%, sugar consumption speed is 4.5g/l/h), ethanol production is all low than SEB4, is respectively 35.93g/l and 32.69g/l.
During 48h, glucose all can consume when 48h by bacterial strain SEB4 and parent strain, and the ethanol production of SEB4 is the highest, is 57.76g/l, and the ethanol production of the sub-RHZ-1 of parent fusion and parent SEB2 is respectively 51.93 and 54.42g/l.
In sum, the fusant RHZ-1 that this research builds with IR-2 and SEB1, and SEB2 is starting strain, the mode merged by protoplasm somatocyte, final acquisition one strain has amphilepsis proterties concurrently, and has the acidproof flocculence Wine brewing yeast strain SEB4 (Lys of outstanding ethanol fermentation performance
+, His
+).
Above-described embodiment is only the preferred embodiments of the present invention, not limiting the scope of the invention, as long as adopt principle of design of the present invention, and the change carried out non-creativeness work on this basis and make, all should belong within protection scope of the present invention.
Claims (4)
1. the strain of a strain acidproof flocculence an industrial strain of S.cerevisiae, is characterized in that, described yeast saccharomyces cerevisiae (
saccharomycescerevisiaesEB4) bacterial strain is preserved in China General Microbiological culture presevation administrative center, and preserving number is CGMCC11324.
2. the construction process of Wine brewing yeast strain as claimed in claim 1, is characterized in that, comprise the following steps:
1) structure of auxotrophic mutation bacterium
1.1) with yeast saccharomyces cerevisiae IR-2 and Saccharomyces Cerevisiae in S EB1 for parent, obtain fusant RHZ-1 by cytogamy mode;
1.2) fusant RHZ-1 and Saccharomyces Cerevisiae in S EB2 is carried out ultraviolet mutagenesis, filter out the single auxotrophic mutation bacterium of fusant RHZ-1 and SEB2 respectively;
1.3) from fusant RHZ-1 single auxotrophic mutation bacterium, the good bacterial strain of flocculence is filtered out, and energy for growth and leavening property evaluation experimental are carried out to this bacterial strain, pick out the auxotrophic mutation bacterium KFU of bacterial strain as subsequent native plast fusion of leavening property optimum; Energy for growth and leavening property evaluation experimental are carried out to SEB2 single auxotrophic mutation bacterium, filters out the auxotrophic mutation bacterium KAGOU of bacterial strain as subsequent native plast fusion of best performance;
2) structure of Protoplast fusant
2.1) using auxotrophic mutation bacterium KFU and auxotrophic mutation bacterium KAGOU as parent, carry out the fusion of protoplasm somatocyte, obtain fusant RHZ-2;
2.2) by the suspension after merging on flat board, screen the fusant RHZ-2 grown, filter out and have flocculence and acid-fast ability and the bacterial strain of leavening property the best, this bacterial strain is Saccharomyces Cerevisiae in S EB4.
3. the construction process of Wine brewing yeast strain according to claim 2, is characterized in that, the specific operation process of described ultraviolet mutagenesis is as follows:
Proceeded to respectively by fusant RHZ-1 and SEB2 in 2%YPD substratum, 30 ° of C activate 16h; Bacterium liquid is applied on YPD flat board after sterilized water dilution, after flat board irradiates 50s under ultraviolet lamp, in 30 ° of C quiescent culture.
4. the construction process of Wine brewing yeast strain according to claim 3, is characterized in that, the screening process of described single auxotrophic mutation bacterium is as follows:
By the bacterial colony photographic reprinting of the YPD slat chain conveyor through ultraviolet lamp process on MM flat board, and in 30 ° of C incubated overnight, the bacterium colony that contrast YPD and MM flat board grows, picking out from YPD flat board cannot at the bacterium colony of MM plated growth, i.e. auxotrophic mutation bacterium;
Add different nutritive substances to MM flat board, each dull and stereotyped a kind of nutrition, cultivates auxotrophic mutation bacterium Simultaneous vaccination, identifies its auxotrophy type, and filter out single auxotrophic mutation bacterium to YPD and MM flat board.
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|---|---|---|---|---|
| CN106244633A (en) * | 2016-08-23 | 2016-12-21 | 广东省生物工程研究所（广州甘蔗糖业研究所） | A kind of reversible regulation and control methods and applications of Yeast Flocculation |
| CN113717874A (en) * | 2021-09-27 | 2021-11-30 | 四川大学 | High-temperature-resistant and high-sugar-resistant saccharomyces cerevisiae strain as well as construction method and application thereof |
| CN116574621A (en) * | 2023-04-23 | 2023-08-11 | 四川大学 | Low-temperature-resistant saccharomyces cerevisiae for mulberry fruit wine and culture method thereof |
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| CN102154138A (en) * | 2011-01-24 | 2011-08-17 | 天津科技大学 | Multi-resistant high-yield alcohol yeast mutant strain TT31 and screening method thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106244633A (en) * | 2016-08-23 | 2016-12-21 | 广东省生物工程研究所（广州甘蔗糖业研究所） | A kind of reversible regulation and control methods and applications of Yeast Flocculation |
| CN106244633B (en) * | 2016-08-23 | 2019-06-18 | 广东省生物工程研究所（广州甘蔗糖业研究所） | A kind of reversible regulation methods and applications of Yeast Flocculation |
| CN113717874A (en) * | 2021-09-27 | 2021-11-30 | 四川大学 | High-temperature-resistant and high-sugar-resistant saccharomyces cerevisiae strain as well as construction method and application thereof |
| CN113717874B (en) * | 2021-09-27 | 2023-04-11 | 四川大学 | High-temperature-resistant and high-sugar-resistant saccharomyces cerevisiae strain as well as construction method and application thereof |
| CN116574621A (en) * | 2023-04-23 | 2023-08-11 | 四川大学 | Low-temperature-resistant saccharomyces cerevisiae for mulberry fruit wine and culture method thereof |
| CN116574621B (en) * | 2023-04-23 | 2024-04-16 | 四川大学 | Low-temperature-resistant saccharomyces cerevisiae for mulberry fruit wine and culture method thereof |
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| CN105368729B (en) | 2019-01-04 |
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