CN102242153A - Method for repeatedly fermenting and utilizing culture medium of pichia pastoris - Google Patents
Method for repeatedly fermenting and utilizing culture medium of pichia pastoris Download PDFInfo
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- CN102242153A CN102242153A CN2011101119917A CN201110111991A CN102242153A CN 102242153 A CN102242153 A CN 102242153A CN 2011101119917 A CN2011101119917 A CN 2011101119917A CN 201110111991 A CN201110111991 A CN 201110111991A CN 102242153 A CN102242153 A CN 102242153A
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Abstract
The invention relates to a method for repeatedly fermenting and utilizing a culture medium of pichia pastoris. The method comprises the following steps of: carrying out batch fermentation, glycerol supplement fermentation, carbon source starvation and methanol supplement fermentation in a single pot with glycerol as a carbon source; after the first fermentation process is completed, taking out a part of bacteria liquid from a fermentation tank and reserving a part of bacteria liquid as seed liquid for repeated fermentation at the same time; and carrying out centrifugal separation on the part of taken bacteria liquid to obtain a yeast paste and supernatant, sterilizing the supernatant and returning the supernatant to the tank, using the returned supernatant as a nutrient solution for repeated fermentation instead of discharging, and repeatedly fermenting and utilizing for several times. The method for repeatedly fermenting and utilizing the culture medium of pichia pastoris has the advantages of improving production efficiency, lowering cost, greatly reducing pollutant discharge and promoting production and application of pichia pastoris because a basic salt solution culture medium used by the pichia pastoris is recycled.
Description
Technical field
The present invention relates to the method that a kind of pichia spp substratum repeats to ferment and utilizes, be applicable to the pichia pastoris phaff to be that starting material are produced yeast extract and utilized pichia pastoris phaff engineering bacteria production purpose product.
Background technology
Traditional pichia spp fermentation process, general mainly form by glycerine batch fermentation stage, glycerine fed-batch fermentation stage and methanol feeding fermentation stage three parts.Wherein, the methanol feeding fermentation stage is a successive process, and from adding methyl alcohol, after through one period long period, thalline just adapts to the methyl alcohol environment gradually, and weight in wet base increases gradually, the later stage reaches peak value to fermentation, and the thalline weight in wet base can reach 350～400g/L.This zymotechnique production cycle was generally about a week, and wherein, growing microorganism speed is the increased logarithmic phase of increased logarithmic phase, glycerine fed-batch fermentation stage and the methanol feeding fermentation stage in glycerine batch fermentation stage faster.Because the glycerine price is higher than methyl alcohol far away, and glycerine do not possess the function of abduction delivering foreign protein, so during the fermentation, glycerine mainly plays the function of rapid propagation thalline.At the methanol induction expression phase, thalline needs the adaptive process to methyl alcohol of segment length's time, thereby influences the throughput rate of whole fermentation process.At present, the pichia spp high density fermentation adopts the basic salt culture medium of high density, the metal ion in the substratum, particularly K usually
+And Mg
2+Content is very high.These metal ions mainly play the thalline osmotic pressure equilibrated effect of keeping during the fermentation, and really the amount that is absorbed by thalline seldom.After fermentation ends, contain a large amount of metal ion solution and be discharged in the environment as waste water, cause very big harm to environment.Simultaneously, remain in the application that metal ion in the pichia spp thalline has also limited the pichia spp tropina to a certain extent.
Summary of the invention
The objective of the invention is to, provide a kind of pichia spp substratum to repeat the method for fermenting and utilizing, the used basic salts solution substratum of pichia spp fermentation is recycled, thereby enhanced productivity, reduce production costs, reduce disposal of pollutants.
Technical scheme of the present invention is: the method that a kind of pichia spp substratum repeats to ferment and utilizes, it comprises the steps:
A, fermentation first: be that carbon source is carried out batch fermentation with glycerine in single jar, pass through the glycerine fed-batch fermentation again, carbon source hunger is carried out the methanol feeding fermentation then;
B, repeat the fermentation:
B1, taking-up part bacterium liquid are used to isolate yeast slurry and supernatant liquor: behind the single batch fermentation end of processing, take out part in the bacterium liquid total amount from fermentor tank and be used for the bacterium liquid that centrifugation goes out yeast slurry and supernatant liquor;
B2, residue bacterium liquid give over to the repetition fermentation seed liquid;
B11, send next procedure with yeast slurry: the bacterium liquid that taking-up is used for separation of supernatant and yeast slurry carries out centrifugation, obtains supernatant liquor and yeast slurry, send next procedure with yeast slurry;
B12, will supernatant liquor return in the fermentor tank after the sterilization, proceeding with methyl alcohol is the fermentation that repeats of carbon source;
Carry out several times by above-mentioned repetition fermentation step and repeat fermentation;
C, last fermentation:
C1, the whole bacterium liquid of the last fermentation of taking-up are used to isolate yeast slurry and waste liquid;
C11, send next procedure with yeast slurry;
C12, waste liquid is discharged, finish whole fermentations.
Further technical scheme is on the basis of technique scheme:
The method that described pichia spp substratum repeats to ferment and utilizes is returned after its supernatant liquor sterilization in the process in the fermentor tank or behind the end of processing, adds aqua sterilisa simultaneously, makes fermentation volume reach initial stocking volume; Stream adds methyl alcohol in repeating fermenting process.
The method that described pichia spp substratum repeats to ferment and utilizes, it repeats in fermenting process by the fermented liquid total amount in the fermentor tank, and the speed that every liter of fermentation broth stream adds methyl alcohol is 5～20 mL/ h.
The method that described pichia spp substratum repeats to ferment and utilizes, the bacterium liquid that is used for separated yeast mud and supernatant liquor is taken out in described fermentation back, be used as the bacterium liquid that next time repeats fermentation seed liquid with staying, the weight percent that accounts for bacterium liquid total amount in the fermentor tank respectively is: take out 60%～90% bacterium liquid, stay 10%～40% bacterium liquid.
The method that described pichia spp substratum repeats to ferment and utilizes repeats to ferment 3～5 times after the fermentation first, again through last fermentation.
The method that described pichia spp substratum repeats to ferment and utilizes repeats to ferment 4 times after the fermentation first, again through last fermentation; The fermentation back is taken out and is used for the bacterium liquid of separated yeast mud and supernatant liquor and stays being used as the bacterium liquid that next time repeats fermentation seed liquid, and the weight percent that accounts for bacterium liquid total amount in the fermentor tank respectively is:
First after the fermentation: take out 85% bacterium liquid, stay 15% bacterium liquid;
The mat woven of fine bamboo strips takes out 80% bacterium liquid after once repeating fermentation, stays 20% bacterium liquid;
Mat woven of fine bamboo strips secondary takes out 75% bacterium liquid after repeating fermentation, stays 25% bacterium liquid;
The mat woven of fine bamboo strips takes out 70% bacterium liquid after repeating fermentation for three times, stays 30% bacterium liquid;
The mat woven of fine bamboo strips takes out 60% bacterium liquid after repeating fermentation for four times, stays 40% bacterium liquid;
After the last fermentation, take out whole bacterium liquid, to the centrifugation of bacterium liquid, send next procedure with yeast slurry again, waste liquid is discharged then, finishes whole fermentations.
The method that described pichia spp substratum repeats to ferment and utilizes repeats to ferment 5 times after the fermentation first, again through last fermentation; The fermentation back is taken out and is used for the bacterium liquid of separated yeast mud and supernatant liquor and stays being used as the bacterium liquid that next time repeats fermentation seed liquid, and the weight percent that at every turn is the bacterium liquid total amount in the fermentor tank respectively is: take out 70% bacterium liquid, stay 30% bacterium liquid.
The method that described pichia spp substratum repeats to ferment and utilizes, described fermentation first, its carbon source starvation time is 1～2h; Stream adds the PTM that contains 10～15mL/L in fermentor tank after carbon source hunger
1The methyl alcohol of solution.
The method that described pichia spp substratum repeats to ferment and utilizes, described single batch fermentation end of processing is that to reach 250～350g/L with the thalline weight in wet base be the concluding time.
The method that described pichia spp substratum repeats to ferment and utilizes, first in the fermenting process, 28.5 ± 0.5 ℃ of controlled temperature, pH5.0 ± 0.1, tank pressure 0.04 MPa, mixing speed 500～900rpm; Fermentation 20～22h, DO rises to 100% rapidly, begins the PTM that stream adds a certain amount of 12mL/L of containing immediately
150% glycerine of solution; Begin stream behind the hungry 1h of carbon source and add the PTM that contains 12mL/L
1The methyl alcohol of solution is set initial flow rate of acceleration 80mL/h, every 2h, feed rate is improved 50%, when feed rate is 270mL/h, keeps this feed rate; In the fermenting process,, then reduce feed rate, come back to more than 20% up to DO if DO is lower than 20%.
To technical scheme of the present invention, principle and outstanding characteristics and effect be:
Briefly, process of the present invention is: through with glycerine being the batch fermentation, fed-batch fermentation of carbon source after the stage, again through carbon source hunger, add then and contain PTM
1The methyl alcohol of solution ferments.Entering substratum after the thalline weight in wet base reaches about 300g/L repeats fermentation and utilizes flow process.After being the single batch fermentation end of processing, stay part bacterium liquid in fermentor tank, as the seed liquor of fermenting next time; With remaining bacterium liquid centrifugation, obtain yeast slurry and supernatant liquor, jar is returned in supernatant liquor sterilization back, rather than discharging, so repeats fermentation and utilizes repeatedly, is generally five times.
Major advantage of the present invention: at traditional methanol feeding fermentation stage, yeast can fast breeding at logarithmic phase, but its adaptive phase and stationary phase rate of propagation all very slow.Substratum circulating fermentation by the present invention's design utilizes flow process, can make the yeast repeated multiple times carry out logarithmic proliferation, the production efficiency height; Simultaneously can recycle the metal-salt substratum, reduce disposal of pollutants.By the centrifugation of bacterium liquid, the supernatant flow processs such as jar of sterilizing back, substratum is repeated fermentation utilization, thereby reduce production costs, enhance productivity, reduce disposal of pollutants.The technology of the present invention's design behind the single batch fermentation end of processing, repeats fermentation with substratum (mainly being metal ion solution) and utilizes repeatedly.Thalline adapted to the methyl alcohol environment owing to stay a jar interior Pichia yeast, so can be bred rapidly at short notice; Simultaneously, owing to the recycling to metal ion solution, the waste liquid that fermentation produces has also reduced a lot.
Description of drawings
Fig. 1 is the inventive method step synoptic diagram;
Fig. 2 is conventional traditional method step synoptic diagram.
Embodiment
In conjunction with the accompanying drawings and embodiments, it is as follows that the invention will be further described:
Embodiment 1
As shown in Figure 1, the method that a kind of pichia spp substratum repeats to ferment and utilizes, it is undertaken by following step:
A, fermentation first: be to be that carbon source is carried out batch fermentation with glycerine in single jar of 50L, pass through the glycerine fed-batch fermentation again, carbon source hunger is carried out the methanol feeding fermentation then; Its carbon source starvation time is 1～2h; After carbon source hunger, in fermentor tank, add the PTM that contains 10～15mL/L
1The methyl alcohol of solution.Present embodiment is first in the fermenting process, 28.5 ± 0.5 ℃ of controlled temperature, pH5.0 ± 0.1, tank pressure 0.04 MPa, mixing speed 500～900rpm; Fermentation 20～22h, DO rises to 100% rapidly, begins the PTM that stream adds a certain amount of 12mL/L of containing immediately
150% glycerine of solution; Begin stream behind the hungry 1h of carbon source and add the PTM that contains 12mL/L
1The methyl alcohol of solution is set initial flow rate of acceleration 80mL/h, every 2h, feed rate is improved 50%, when feed rate is 270mL/h, keeps this feed rate; In the fermenting process,, then reduce feed rate, come back to more than 20% up to DO if DO is lower than 20%.Described single batch fermentation end of processing is that to reach 250～350g/L with the thalline weight in wet base be the concluding time point.Present embodiment is that to reach 300g/L with the thalline weight in wet base be the concluding time point, when promptly the thalline weight in wet base reaches 300g/L, stops fermentation;
B, repeat the fermentation:
B1, taking-up part bacterium liquid are used to isolate yeast slurry and supernatant liquor: behind the single batch fermentation end of processing, take out part in the bacterium liquid total amount from fermentor tank and be used for the bacterium liquid that centrifugation goes out yeast slurry and supernatant liquor;
B2, residue bacterium liquid give over to the repetition fermentation seed liquid;
B11, send next procedure with yeast slurry: the bacterium liquid that taking-up is used for separation of supernatant and yeast slurry carries out centrifugation, obtains supernatant liquor and yeast slurry, send next procedure with yeast slurry;
B12, will supernatant liquor return in the fermentor tank after the sterilization, proceeding with methyl alcohol is the fermentation that repeats of carbon source; Supernatant liquor is to use as the nutritive medium that repeats to ferment, rather than discharging;
Carry out several times by above-mentioned repetition fermentation step and repeat fermentation;
C, last fermentation:
C1, the whole bacterium liquid of the last fermentation of taking-up are used to isolate yeast slurry and waste liquid;
C11, send next procedure with yeast slurry;
C12, waste liquid is discharged, finish whole fermentations.
After the supernatant liquor sterilization, return in the process in the fermentor tank or behind the end of processing, add aqua sterilisa simultaneously, make fermentation volume reach initial stocking volume; Stream adds methyl alcohol in repeating fermenting process.Repeat in the fermenting process by the fermented liquid total amount in the fermentor tank, the speed that every liter of fermentation broth stream adds methyl alcohol is 5～20 mL/ h, and present embodiment adopts 15 mL/ h.Described fermentation back is taken out and is used for the bacterium liquid of separated yeast mud and supernatant liquor and stays being used as the bacterium liquid that next time repeats fermentation seed liquid, and the weight percent that accounts for bacterium liquid total amount in the fermentor tank respectively is: take out 60%～90% bacterium liquid, stay 10%～40% bacterium liquid.Repeat to ferment 3～5 times after the fermentation first, again through last fermentation.After fermenting first, present embodiment repeats to ferment 4 times, again through last fermentation; The fermentation back is taken out and is used for the bacterium liquid of separated yeast mud and supernatant liquor and stays being used as the bacterium liquid that next time repeats fermentation seed liquid, and the weight percent that accounts for bacterium liquid total amount in the fermentor tank respectively is:
First after the fermentation: take out 85% bacterium liquid, stay 15% bacterium liquid;
The mat woven of fine bamboo strips takes out 80% bacterium liquid after once repeating fermentation, stays 20% bacterium liquid;
Mat woven of fine bamboo strips secondary takes out 75% bacterium liquid after repeating fermentation, stays 25% bacterium liquid;
The mat woven of fine bamboo strips takes out 70% bacterium liquid after repeating fermentation for three times, stays 30% bacterium liquid;
The mat woven of fine bamboo strips takes out 60% bacterium liquid after repeating fermentation for four times, stays 40% bacterium liquid;
After the last fermentation, take out whole bacterium liquid, to the centrifugation of bacterium liquid, send next procedure with yeast slurry again, waste liquid is discharged then, finishes whole fermentations.
Described single batch fermentation end of processing is that to reach 250～350g/L with the thalline weight in wet base be the end of processing time.Present embodiment fermentation total time 160h can obtain about 9kg dry yeast altogether.The metal ion waste liquid amount of discharging has reduced 70% than common method shown in Figure 2.
Embodiment 2:Different with the foregoing description 1 is: repeat to ferment 5 times after the fermentation first, again through last fermentation; The fermentation back is taken out and is used for the bacterium liquid of separated yeast mud and supernatant liquor and stays being used as the bacterium liquid that next time repeats fermentation seed liquid, and the weight percent that at every turn is the bacterium liquid total amount in the fermentor tank respectively is: take out 70% bacterium liquid, stay 30% bacterium liquid.
Repeat in the fermenting process by the fermented liquid total amount in the fermentor tank, the speed that every liter of fermentation broth stream adds methyl alcohol is 20 mL/ h.Present embodiment fermentation total time 160h obtains about 8kg dry yeast.The metal ion waste liquid amount of discharging has reduced 65% than common method shown in Figure 2.
Claim protection domain of the present invention is not limited to the foregoing description.
Claims (10)
1. the method that the pichia spp substratum repeats to ferment and utilize is characterized in that it comprises the steps:
A, fermentation first: be that carbon source is carried out batch fermentation with glycerine in single jar, pass through the glycerine fed-batch fermentation again, carbon source hunger is carried out the methanol feeding fermentation then;
B, repeat the fermentation:
B1, taking-up part bacterium liquid are used to isolate yeast slurry and supernatant liquor: behind the single batch fermentation end of processing, take out part in the bacterium liquid total amount from fermentor tank and be used for the bacterium liquid that centrifugation goes out yeast slurry and supernatant liquor;
B2, residue bacterium liquid give over to the repetition fermentation seed liquid;
B11, send next procedure with yeast slurry: the bacterium liquid that taking-up is used for separation of supernatant and yeast slurry carries out centrifugation, obtains supernatant liquor and yeast slurry, send next procedure with yeast slurry;
B12, will supernatant liquor return in the fermentor tank after the sterilization, proceeding with methyl alcohol is the fermentation that repeats of carbon source;
Carry out several times by above-mentioned repetition fermentation step and repeat fermentation;
C, last fermentation:
C1, the whole bacterium liquid of the last fermentation of taking-up are used to isolate yeast slurry and waste liquid;
C11, send next procedure with yeast slurry;
C12, waste liquid is discharged, finish whole fermentations.
2. the method that pichia spp substratum according to claim 1 repeats to ferment and utilizes is characterized in that, returns after the supernatant liquor sterilization in the process in the fermentor tank or behind the end of processing, adds aqua sterilisa simultaneously, makes fermentation volume reach initial stocking volume; Stream adds methyl alcohol in repeating fermenting process.
3. the method that pichia spp substratum according to claim 2 repeats to ferment and utilizes is characterized in that, repeats in the fermenting process by the fermented liquid total amount in the fermentor tank, and the speed that every liter of fermentation broth stream adds methyl alcohol is 5～20 mL/ h.
4. repeat the method for fermenting and utilizing according to claim 1 or 2 or 3 described pichia spp substratum, it is characterized in that, the bacterium liquid that is used for separated yeast mud and supernatant liquor is taken out in described fermentation back, be used as the bacterium liquid that next time repeats fermentation seed liquid with staying, the weight percent that accounts for bacterium liquid total amount in the fermentor tank respectively is: take out 60%～90% bacterium liquid, stay 10%～40% bacterium liquid.
5. the method that pichia spp substratum according to claim 4 repeats to ferment and utilizes is characterized in that, repeats to ferment 3～5 times after the fermentation first, again through last fermentation.
6. the method that pichia spp substratum according to claim 5 repeats to ferment and utilizes is characterized in that, repeats to ferment 4 times after the fermentation first, again through last fermentation; The fermentation back is taken out and is used for the bacterium liquid of separated yeast mud and supernatant liquor and stays being used as the bacterium liquid that next time repeats fermentation seed liquid, and the weight percent that accounts for bacterium liquid total amount in the fermentor tank respectively is:
First after the fermentation: take out 85% bacterium liquid, stay 15% bacterium liquid;
The mat woven of fine bamboo strips takes out 80% bacterium liquid after once repeating fermentation, stays 20% bacterium liquid;
Mat woven of fine bamboo strips secondary takes out 75% bacterium liquid after repeating fermentation, stays 25% bacterium liquid;
The mat woven of fine bamboo strips takes out 70% bacterium liquid after repeating fermentation for three times, stays 30% bacterium liquid;
The mat woven of fine bamboo strips takes out 60% bacterium liquid after repeating fermentation for four times, stays 40% bacterium liquid;
After the last fermentation, take out whole bacterium liquid, to the centrifugation of bacterium liquid, send next procedure with yeast slurry again, waste liquid is discharged then, finishes whole fermentations.
7. the method that pichia spp substratum according to claim 5 repeats to ferment and utilizes is characterized in that, repeats to ferment 5 times after the fermentation first, again through last fermentation; The fermentation back is taken out and is used for the bacterium liquid of separated yeast mud and supernatant liquor and stays being used as the bacterium liquid that next time repeats fermentation seed liquid, and the weight percent that at every turn is the bacterium liquid total amount in the fermentor tank respectively is: take out 70% bacterium liquid, stay 30% bacterium liquid.
8. repeat the method for fermenting and utilizing according to claim 1 or 2 or 3 described pichia spp substratum, it is characterized in that, described fermentation first, its carbon source starvation time is 1～2h; Stream adds the PTM that contains 10～15mL/L in fermentor tank after carbon source hunger
1The methyl alcohol of solution.
9. repeat the method for fermenting and utilizing according to claim 1 or 2 or 3 described pichia spp substratum, it is characterized in that, described single batch fermentation end of processing is that to reach 250～350g/L with the thalline weight in wet base be the concluding time.
10. the method that pichia spp substratum according to claim 6 repeats to ferment and utilizes is characterized in that, first in the fermenting process, and 28.5 ± 0.5 ℃ of controlled temperature, pH5.0 ± 0.1, tank pressure 0.04 MPa, mixing speed 500～900rpm; Fermentation 20～22h, DO rises to 100% rapidly, begins the PTM that stream adds a certain amount of 12mL/L of containing immediately
150% glycerine of solution; Begin stream behind the hungry 1h of carbon source and add the PTM that contains 12mL/L
1The methyl alcohol of solution is set initial flow rate of acceleration 80mL/h, every 2h, feed rate is improved 50%, when feed rate is 270mL/h, keeps this feed rate; In the fermenting process,, then reduce feed rate, come back to more than 20% up to DO if DO is lower than 20%.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105200101A (en) * | 2015-10-19 | 2015-12-30 | 华中农业大学 | Method for repeated fermentation of Pichia pastoris for expression of foreign proteins |
| CN112048450A (en) * | 2020-08-14 | 2020-12-08 | 润盈生物工程（上海）有限公司 | Method for repeatedly fermenting twice by using bacillus coagulans fermentation centrifugal supernatant |
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| CN1281051A (en) * | 1999-07-19 | 2001-01-24 | 天津轻工业学院 | Process for preparing bacterial cellulose by glucose oxidation and bacillus fermentation |
| WO2009006909A1 (en) * | 2007-07-10 | 2009-01-15 | Nordbiochem OÜ | Process for producing organic acids by continuous fermentation |
| CN102010833A (en) * | 2010-06-21 | 2011-04-13 | 唐传生物科技（厦门）有限公司 | Novel pichia strain and method for mixing and culturing same to biologically detoxify hemicellulose hydrolysate |
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2011
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1057071A (en) * | 1991-07-18 | 1991-12-18 | 中国科学院上海有机化学研究所 | Pollution-free preparation of corn yeast |
| CN1281051A (en) * | 1999-07-19 | 2001-01-24 | 天津轻工业学院 | Process for preparing bacterial cellulose by glucose oxidation and bacillus fermentation |
| WO2009006909A1 (en) * | 2007-07-10 | 2009-01-15 | Nordbiochem OÜ | Process for producing organic acids by continuous fermentation |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105200101A (en) * | 2015-10-19 | 2015-12-30 | 华中农业大学 | Method for repeated fermentation of Pichia pastoris for expression of foreign proteins |
| CN105200101B (en) * | 2015-10-19 | 2019-04-16 | 华中农业大学 | A kind of method that Pichia yeast weight answers fermentation expression foreign protein |
| CN112048450A (en) * | 2020-08-14 | 2020-12-08 | 润盈生物工程（上海）有限公司 | Method for repeatedly fermenting twice by using bacillus coagulans fermentation centrifugal supernatant |
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