CN105112496A - Method for monitoring fermentation end-point of pichia pastoris - Google Patents
Method for monitoring fermentation end-point of pichia pastoris Download PDFInfo
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Abstract
The invention relates to the field of pichia pastoris expression foreign protein, in particular to a method for monitoring fermentation end-point in a process of using pichia pastoris induced expression foreign protein. The method includes the following steps: (1), regularly sampling fermentation liquid; (2), treating fermentation liquid samples; (3), measuring bacterial concentration and conductivity of treated samples respectively; (4), monitoring changes of bacterial concentration and conductivity of the fermentation liquid along fermentation time, and determining the fermentation end-point when the bacterial concentration reaches a highest point and the conductivity is lowered to 28-35mS/cm. The problems of time consumption, labor consumption and high cost when a high performance chromatography method is used to judge the fermentation end-point are avoided, and the method capable of judging the fermentation end-point of pichia pastoris quickly and is simple, practical and capable of lowering cost and improving production efficiency.
Description
Technical field
The present invention relates to Pichia anomala expression foreign protein field, particularly use the method for monitoring fermentation termination in pichia spp abduction delivering foreign protein technique.
Background technology
Pichia spp is the desirable expression system (CereghinoGP of multiple protein, CereghinoJL, IlgenC, CreggJM.Productionofrecombinantproteinsinfermentercultur esoftheyeastPichiapastoris.CurrOpinBiotechnol.2002, 13 (4): 329 – 332), it is when fermentation cylinder for fermentation is cultivated, generally be divided into three phases (Invitrogenlifetechnologies company, Pichiafermentationprocessguidelines): first utilize basic salts solution, PTM1 trace element carries out batch fermentation cultivation as initial medium, then until batch fermentation be cultured to glycerine exhaust completely time, carry out glycerol batch feed-batch culture, finally after glycerol batch feed-batch culture terminates, carry out methyl alcohol Fed batch fementation, i.e. the protein production stage.Wherein, in the later stage in protein production stage, the proteolytic ferment discharged when thalline is dead can protein degradation, causes fermentation yield to reduce, and therefore, fermentation termination terminates to ferment most important to obtaining high protein output in time to select suitable method to judge.
At present, judge that fermentation termination generally adopts high performance liquid chromatography or empirical method.High performance liquid chromatography can detect the content of target protein in fermented liquid exactly to judge fermentation termination (CN104297378A), but, this method needs special instrument, buy and running cost higher, and equipment setup time and detection time longer, to be checked measure result after, often miss and most suitablely put the tank time, portioned product is caused to be degraded, extend manufacture cycle, so not only cannot obtain high yield, also add foreign matter content and follow-up purifying process cost.Empirical method is fermentation termination determination methods conventional in producing, on specific process conditions, how series-produced basis, namely terminate after fermentation culture to certain hour to ferment (CN101348820B), and for pichia spp fermentation, usually terminate (the Liu Haifeng that ferments when the dense increase of bacterium is slow.The technical study of Recombulin precursor conversion adult islet element and insulin detemir.East China University of Science Ph.D. Dissertation.2013), but this empirical method accurately cannot judge fermentation termination, usually causes in advance or delay terminating fermentation, causes damage to production.
Summary of the invention
The object of this invention is to provide a kind of method of monitoring pichia spp fermentation termination, solve prior art and fast, accurately cannot judge pichia spp fermentation termination and reduce the problem of purifying cost.
The present invention is dense by the bacterium of mensuration fermented liquid and conductivity indices combines the fermentation termination judging pichia spp, and concrete steps are:
(1) timing sampling of fermented liquid: abduction delivering starts regularly to get fermentation broth sample continuously afterwards; Generally within every 12 hours after abduction delivering starts, get fermentation broth sample once during the fermentation, after abduction delivering 36-48h, increase sampling frequency, every 1-6h sampling once.
(2) process of fermentation broth sample: the difference according to institute's testing index carries out centrifugal or dilution process to sample;
(3) testing index: the dense and specific conductivity of the sample determination bacterium processed, and draw the change curve of the dense and specific conductivity of bacterium with induction time;
(4) judgement of fermentation termination: the dense and specific conductivity of monitoring fermented liquid bacterium with the change of fermentation time, when the dense index of bacterium peak, specific conductivity is fermentation termination when being reduced to 28 ~ 35mS/cm.
The dense index of the bacterium measured in aforesaid method is thalline weight in wet base, dry cell weight, OD
600in one, need to get when measuring thalline weight in wet base fermentation broth sample 1 ~ 5ml be placed in the centrifuge tube of weighing in advance in centrifugal 5 ~ 10 minutes of 3000 ~ 12000rpm and the pre-treatment that supernatant discarded carries out sample prepare; When measuring bacterium dense middle dry cell weight, sample treatment is: get fermentation broth sample 1 ~ 5ml and be placed in the centrifuge tube of weighing in advance in 3000 ~ 12000rpm centrifugal 5 ~ 10 minutes, supernatant discarded, and be placed in 80 DEG C of vacuum-dryings to constant weight; Measure the dense middle OD of bacterium
600the treatment process of sample be: by fermentation broth sample dilute with water 100 ~ 1000 times, make final reading between 0.2 ~ 0.8;
The treatment process measuring sample during specific conductivity is: get fermentation broth sample 1 ~ 5ml in centrifuge tube, centrifugal 5 ~ 10 minutes of 3000 ~ 12000rpm.
In preferred step (3) specific conductivity mensuration in get centrifugal after sample pure water dilution 5 ~ 10 times after detect specific conductivity.
Dense and the specific conductivity of monitoring fermented liquid bacterium is with the change of induction time, and when the dense increase of bacterium slowly close to vertex or start after peaking to decline, specific conductivity is reduced to 28 ~ 35mS/cm simultaneously, is fermentation termination.
Preferably, when the dense increase of bacterium slowly close to vertex or start after peaking to decline, simultaneously specific conductivity is reduced to 30 ~ 32mS/cm, is fermentation termination.
Pichia spp fermentation in the application is the process utilizing pichia spp to carry out methanol induction expression foreign protein in fermentor tank.The protein production stage in pichia spp fermenting process, thalline is while utilize methyl alcohol to produce target protein, slowly growth causes the dense increase of bacterium on one side, and in pichia spp fermentation, salt in substratum is except maintaining osmotic balance inside and outside thalline, also will meet the needs of thalli growth, metabolism, so salt concn reduces along with the carrying out of fermentation, macro manifestations is the reduction of specific conductivity.
In addition due to phase after fermentation, thalline reproduction speed lowers, dead thalline increases gradually, while causing that bacterium is dense and peaking gradually, the proteolytic enzyme protolysate that thalline death discharges causes object product to reduce, when the protein content of increase and the protein content of minimizing reach balance, show as output and reach the highest, and in this stage, the death of thalline is except discharging proteolytic enzyme, electrolyte inside cell, as salt, organic acid etc., also fermented liquid is discharged into, increase the electroconductibility of fermented liquid, the electroconductibility that partial offset thalli growth metabolic exhaustion salt causes declines, show as specific conductivity decline and become slow.When occur specific conductivity decline slow phenomenon time, mean that expression amount is close to high point, through experiment prove, when the dense increase of bacterium slowly close to vertex or start after peaking decline and specific conductivity is reduced to 28 ~ 35mS/cm, be preferably 30 ~ 32mS/cm, be fermentation termination.
Beneficial effect of the present invention is:
(1) in the method, employing mensuration bacterium is dense judges reaction end with method that is specific conductivity, and compare existing HPLC method, the pre-treatment of sample is simple, easy handling, detection time is short, is convenient to terminate fermentation in time, decrease fermentation energy consumption and time, increase work efficiency, reduce production cost.
(2) the method employing bacterium is dense judges fermentation termination with dual indexes that is specific conductivity, compares empirical method, more can judge reaction end exactly, avoids in advance or delays terminating fermentation, improve production efficiency.
Accompanying drawing explanation
Fig. 1 is the graphic representation that when pichia spp recombinant bacterium expresses foreign protein in BSM substratum in embodiment 1, specific conductivity, thalline weight in wet base and fermented liquid supernatant target protein content change with induction time.-◆-be conductivity variations curve ,-▇-be thalline weight in wet base change curve ,-▲-be protein content change curve.
Fig. 2 is the graphic representation that when pichia spp recombinant bacterium expresses foreign protein in BSM substratum in embodiment 2, specific conductivity, dry cell weight and fermented liquid supernatant target protein content change with induction time.-◆-be conductivity variations curve ,-▇-be dry cell weight change curve ,-▲-be protein content change curve.
Fig. 3 is specific conductivity, OD when pichia spp recombinant bacterium expresses foreign protein in BSM substratum in embodiment 3
600and the graphic representation that fermented liquid supernatant target protein content changes with induction time.-◆-be conductivity variations curve ,-▇-be OD
600change curve ,-▲-be protein content change curve.
Embodiment
Explain the present invention further below by way of specific embodiment, embodiment does not only limit the present invention's scope required for protection for illustration of the present invention.
Embodiment 1:
The fermentation culture of Pichia anomala expression foreign protein is carried out, inducing temperature 28 DEG C, induction pH5.0 according to the substratum provided in Invitrogenlifetechnologies company " Pichiafermentationprocessguidelines " and method.Carry out according to the following step in the process expressed:
(1) timing sampling of fermented liquid: after abduction delivering starts, every 12h is aseptic gets fermentation broth sample once; After induction 48h, every 3h gets fermentation broth sample once;
(2) process of fermentation broth sample: get 2 5ml centrifuge tubes, respectively with scales/electronic balance weighing and record; Get fermentation broth sample 4ml respectively and be placed in centrifuge tube, the centrifugal 10min of 3000rpm; Get supernatant liquor 1ml, pure water is diluted to 5ml, specific conductivity to be measured, and all the other supernatant liquors are stored in 4 DEG C of refrigerator stand-by high performance liquid chromatography testing goal protein contents, removes the centrifuge tube thalline weight in wet base to be measured of supernatant liquor.
(3) thalline weight in wet base and specific conductivity is measured: the centrifuge tube removing supernatant liquor is weighed, and calculates thalline weight in wet base; The fermented liquid supernatant of having diluted measures specific conductivity; Make thalline weight in wet base, specific conductivity with the change curve (Fig. 1) of induction time.
(4) judgement of fermentation termination: the dense and specific conductivity of monitoring fermented liquid bacterium is with the change of fermentation time.Abduction delivering 48h, sample as stated above and measure specific conductivity, thalline weight in wet base is respectively 32.63mS/cm and 0.527g/ml; During induction 51h, sample and record specific conductivity and thalline weight in wet base is respectively 31.0mS/cm and 0.528g/ml, compare with 48h, thalline weight in wet base does not almost increase, and specific conductivity is close to 30mS/cm, and judgement reaches fermentation termination.
For checking the validity of present method, not terminating fermentation, proceeding to cultivate, until specific conductivity lower than 30mS/cm and thalline weight in wet base start to reduce.The supernatant samples high performance liquid chromatography left and taken is detected, result display (Fig. 1), the front 48h after induction starts, in fermented liquid supernatant liquid, target protein content increases fast, during induction 51h, protein content reaches maximum 950mg/L, and thereafter, protein content significantly reduces.Really for fermentation termination when which illustrating induction 51h, above-mentionedly utilize the dense and specific conductivity of fermented liquid bacterium to combine to judge that the method for pichia spp fermentation termination is effective.
Embodiment 2:
The fermentation culture of Pichia anomala expression foreign protein is carried out, inducing temperature 25 DEG C, induction pH5.0 according to the substratum provided in Invitrogenlifetechnologies company " Pichiafermentationprocessguidelines " and method.Carry out according to the following step in the process expressed:
(1) timing sampling of fermented liquid: after abduction delivering starts, every 12h is aseptic gets fermentation broth sample once; After induction 48h, every 3h gets fermentation broth sample once;
(2) process of fermentation broth sample: get 5ml centrifuge tube 2, respectively with scales/electronic balance weighing and record; Get fermentation broth sample 4ml respectively and be placed in centrifuge tube, the centrifugal 5min of 12000rpm.Get supernatant liquor 1ml, pure water is diluted to 5ml, specific conductivity to be measured; All the other supernatant liquors are stored in 4 DEG C of refrigerator stand-by high performance liquid chromatography testing goal protein contents.Remove the centrifuge tube of supernatant liquor, be placed in 80 DEG C of vacuum-dryings, dry cell weight to be measured.
(3) dry cell weight and specific conductivity is measured: vacuum-drying, to the centrifuge tube with thalline of constant weight, in conjunction with the centrifuge tube weight of record, calculates dry cell weight; The fermented liquid supernatant of having diluted measures specific conductivity; Make dry cell weight, specific conductivity with the change curve (Fig. 2) of induction time.
(4) judgement of fermentation termination: the dense and specific conductivity of monitoring fermented liquid bacterium is with the change of fermentation time.Abduction delivering 48h, sample and measure specific conductivity, dry cell weight is respectively 32.64mS/cm and 119.1g/L.During induction 51h, sample and record specific conductivity and dry cell weight is respectively 31.60mS/cm and 125.2g/L.During induction 54h, sample and record specific conductivity and dry cell weight is respectively 30.24mS/cm and 128.4g/L.Compare with 51h, dry cell weight increases slowly, and specific conductivity is close to 30mS/cm, judges to reach fermentation termination.
For checking the validity of present method, proceed to cultivate, until specific conductivity lower than 30mS/cm and dry cell weight start to reduce.The supernatant samples high performance liquid chromatography left and taken is detected, during result display induction 54h, the protein content of fermented supernatant fluid reaches maximum 1022mg/L, thereafter protein content obviously reduces, and the dry cell weight still slowly increase (Fig. 2) that this stage is corresponding, if this illustrates that empirically method only adopts the dense judge index as fermentation ends of bacterium, the most suitable end time will be missed, utilize the dense and specific conductivity of fermented liquid bacterium to combine the method judging pichia spp fermentation termination more effective.
Embodiment 3:
The fermentation culture of Pichia anomala expression foreign protein is carried out, inducing temperature 25 DEG C, induction pH5.0 according to the substratum provided in Invitrogenlifetechnologies company " Pichiafermentationprocessguidelines " and method.Carry out according to the following step in the process expressed:
(1) timing sampling of fermented liquid: after abduction delivering starts, every 12h is aseptic gets fermentation broth sample once; After induction 48h, every 3h gets fermentation broth sample once;
(2) process of fermentation broth sample: get 1ml fermentation broth sample in 15ml test tube, with water stepwise dilution to 1000 times, OD to be measured
600; Get 5ml centrifuge tube 2, get fermentation broth sample 4ml respectively and be placed in centrifuge tube, the centrifugal 5min of 10000rpm, get supernatant liquor 1ml, pure water is diluted to 5ml, specific conductivity to be measured, and all the other supernatant liquors are stored in 4 DEG C of refrigerator stand-by high performance liquid chromatography testing goal protein contents.
(3) OD is measured
600and specific conductivity: the sample handled well measures OD respectively
600and specific conductivity, make the change curve (Fig. 3) with induction time.
(4) judgement of fermentation termination: the dense and specific conductivity of monitoring fermented liquid bacterium is with the change of fermentation time.Abduction delivering 48h, samples and measures specific conductivity, OD
600be respectively 32.76mS/cm and 598.Induction 51h, samples and records specific conductivity and OD
600be respectively 31.7mS/cm and 605.During induction 54h, specific conductivity and OD
600be respectively 30.52mS/cm and 610g/ml.Compare with 51h, OD
600increase slowly, and specific conductivity is close to 30mS/cm, judges that expression amount reaches high point, fermentation should be terminated.
For checking the validity of present method, continue to cultivate, until specific conductivity is lower than 30mS/cm and OD
600start to reduce.The supernatant samples high performance liquid chromatography left and taken detected, during result display induction 54h, the protein content of fermented supernatant fluid reaches maximum 902mg/L, and protein content obviously reduces thereafter, and the OD that this stage is corresponding
600still slowly (Fig. 3) is increased, if this illustrates that empirically method only adopts the dense judge index as fermentation ends of bacterium, the most suitable end time will be missed, utilize the dense and specific conductivity of fermented liquid bacterium to combine the method judging pichia spp fermentation termination more effective.
Claims (9)
1. monitor a method for pichia spp fermentation termination, it is characterized in that, the bacterium of utilization mensuration fermented liquid is dense combines with conductivity indices the fermentation termination judging pichia spp, and it comprises the following steps:
(1) timing sampling of fermented liquid: abduction delivering starts regularly to get fermentation broth sample continuously afterwards;
(2) process of fermentation broth sample: the difference according to institute's testing index carries out centrifugal or dilution process to sample;
(3) testing index: the dense and specific conductivity of the sample determination bacterium processed, and draw the change curve of the dense and specific conductivity of bacterium with induction time;
(4) judgement of fermentation termination: the dense and specific conductivity of monitoring fermented liquid bacterium with the change of fermentation time, when the dense index of bacterium peak, specific conductivity is fermentation termination when being reduced to 28 ~ 35mS/cm.
2. a kind of method of monitoring pichia spp fermentation termination according to claim 1, is characterized in that, the described dense index of bacterium needing mensuration is thalline weight in wet base, dry cell weight, OD
600in one.
3. a kind of method of monitoring pichia spp fermentation termination according to claim 1, it is characterized in that, in step (1), timing serial sampling is for get fermentation broth sample once in every 12 hours after abduction delivering starts during the fermentation, after abduction delivering 36-48h, increase sampling frequency, every 1-6h sampling once.
4. a kind of method of monitoring pichia spp fermentation termination according to claim 1 and 2, is characterized in that, in step (2), the treatment process of sample is:
(1) sample treatment measuring bacterium dense middle thalline weight in wet base is: get fermentation broth sample 1 ~ 5ml and be placed in the centrifuge tube of weighing in advance in 3000 ~ 12000rpm centrifugal 5 ~ 10 minutes, supernatant discarded;
(2) sample treatment of bacterium dense middle dry cell weight is measured: get fermentation broth sample 1 ~ 5ml and be placed in the centrifuge tube of weighing in advance in 3000 ~ 12000rpm centrifugal 5 ~ 10 minutes, supernatant discarded, and be placed in 80 DEG C of vacuum-dryings to constant weight;
(3) the dense middle OD of bacterium is measured
600sample treatment: by fermentation broth sample dilute with water 100 ~ 1000 times, make final reading between 0.2 ~ 0.8.
5. a kind of method of monitoring pichia spp fermentation termination according to claim 1, it is characterized in that, measuring conductivity sample treatment process in step (2) is: get fermentation broth sample 1 ~ 5ml in centrifuge tube, centrifugal 5 ~ 10 minutes of 3000 ~ 12000rpm.
6. a kind of method of monitoring pichia spp fermentation termination according to claim 1, is characterized in that, the mensuration of specific conductivity in step (3), it is characterized in that centrifugal after sample pure water dilution 5 ~ 10 times after detect specific conductivity.
7. a kind of method of monitoring pichia spp fermentation termination according to claim 1, it is characterized in that, the determination methods of fermentation termination is in step (4): the dense and specific conductivity of monitoring fermented liquid bacterium is with the change of induction time, when the dense increase of bacterium slowly close to vertex or start after peaking decline, specific conductivity is reduced to 28 ~ 35mS/cm simultaneously, is fermentation termination.
8. a kind of method of monitoring pichia spp fermentation termination according to any one of claim 1 or 7, it is characterized in that, when the dense increase of bacterium slowly close to vertex or start after peaking to decline, simultaneously specific conductivity is reduced to 30 ~ 32mS/cm, is fermentation termination.
9. a kind of method of monitoring pichia spp fermentation termination according to claim 1, is characterized in that, pichia spp fermentation is the process utilizing pichia spp to carry out methanol induction expression foreign protein in fermentor tank.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106370603A (en) * | 2016-09-05 | 2017-02-01 | 安徽绿之鑫信息科技有限责任公司 | Method for judging hairy tofu fermentation terminal point by means of light sensor |
| CN108546637A (en) * | 2018-05-23 | 2018-09-18 | 华东理工大学 | Restructured Pichia pastoris in expression glucose oxidase installation for fermenting and fermentation culture method |
| CN108624516A (en) * | 2017-03-20 | 2018-10-09 | 华东理工大学 | A kind of metabolite amount in raising fermented cells and the method for preparing IDMS standard items |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106370603A (en) * | 2016-09-05 | 2017-02-01 | 安徽绿之鑫信息科技有限责任公司 | Method for judging hairy tofu fermentation terminal point by means of light sensor |
| CN106370603B (en) * | 2016-09-05 | 2018-10-12 | 安徽绿之鑫信息科技有限责任公司 | A method of judging bean curd of fermented fermentation termination using light sensor |
| CN108624516A (en) * | 2017-03-20 | 2018-10-09 | 华东理工大学 | A kind of metabolite amount in raising fermented cells and the method for preparing IDMS standard items |
| CN108546637A (en) * | 2018-05-23 | 2018-09-18 | 华东理工大学 | Restructured Pichia pastoris in expression glucose oxidase installation for fermenting and fermentation culture method |
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